Collaborative studies to establish the first World Health Organization International Standard for detection of human antibody against human platelet antigen-3a

BACKGROUND AND OBJECTIVES: The platelet-specific antibody anti-human platelet antigen-3a (anti-HPA-3a) is involved in neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet refractoriness. However, HPA-3a antibodies are often difficult to detect, probably because the antigen is labile. This report describes the production of a freeze-dried preparation of pooled human plasma, coded 03/190, containing IgG antibodies against the HPA-3a. The material is intended for use as a minimum sensitivity reagent in glycoprotein-specific assays currently used for anti-HPA-3a detection. Laboratories can use it to assess the sensitivity of their 'in-house' assays for anti-HPA-3a and to calibrate local controls for routine use in each batch of tests. MATERIALS AND METHODS: Plasma containing anti-HPA-3a was obtained from a mother of two babies both born with severe thrombocytopenia, and following dilution it was freeze dried in glass ampoules. RESULTS: Two collaborative studies demonstrated that the candidate material contained anti-HPA-3a and human leucocyte antigen (HLA) class I antibodies, but no other HPA antibodies that might confuse the detection of the anti-HPA-3a. The minimum dilution that should give a positive result was determined to be 1 : 8 by two further international collaborative studies involving a total of 49 laboratories in 23 countries. CONCLUSION: The material also contains HLA antibodies and is suitable for use only in techniques that are glycoprotein specific (i.e. monoclonal antibody immobilization of platelet antigens and enzyme-linked immunosorbent assay) where only HPA antibodies will be detected. This standard will allow laboratories to measure their sensitivity of detection of anti-HPA-3a and will also allow those laboratories with relatively insensitive techniques to monitor their performance as they improve their methodology.     

Collaborative studies to establish the first WHO Reference Reagent for detection of human antibody against human platelet antigen-5b

BACKGROUND AND OBJECTIVES: This report describes the production of a freeze-dried preparation of pooled human plasma, coded 99/666, containing immunoglobulin G (IgG) antibodies against human platelet antigen 5b (HPA-5b). MATERIALS AND METHODS: The material is intended for use as a minimum sensitivity reagent in the assays currently used for detection of antibodies to HPA-5b. Laboratories can use it to assess the sensitivity of their 'in-house' assays for antibodies to HPA-5b and to calibrate local controls for routine use in each batch of tests. RESULTS: Two collaborative studies demonstrated that the two candidate materials contained antibodies to HPA-5b and that there were no other HPA or human leucocyte antigen (HLA) antibodies which might confuse the detection of antibodies to HPA-5b. The two samples were pooled and freeze-dried in 1-ml ampoules. CONCLUSIONS: The minimum dilution of the antibody against 5b required to yield a positive result was determined, by two international collaborative studies involving a total of 49 laboratories in 26 countries, to be 1 in 2.     

A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

Background

Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays.

Design and Methods

Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a.

Results

This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies.

Conclusions

This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes.     

Collaborative study to establish the first international standard for quantitation of anti-HPA-1a

BACKGROUND AND OBJECTIVES: This report describes the production of a freeze-dried preparation of pooled human plasma, containing immunoglobulin G (IgG) antibodies against the human platelet antigen 1a (HPA-1a). The material, coded 03/152, is proposed as an International Standard containing 100 arbitrary units of anti-HPA-1a for use in quantitative assays to determine the anti-HPA-1a activity in clinical samples. MATERIALS AND METHODS: Plasma samples containing potent anti-HPA-1a were pooled and freeze dried in 1-ml ampoules. In addition, three individual plasma samples were selected which had varying levels of anti-HPA-1a activity. The anti-HPA-1a activity of these three samples was determined by using a variety of quantitative assays with the proposed standard as a reference. RESULTS: An international collaborative study, which was part of the 2004 ISBT Platelet Immunology Workshop, involved 39 laboratories in 24 countries and showed that the anti-HPA-1a activity in three test samples could be reliably determined by using the proposed standard. CONCLUSIONS: Laboratories can use this standard to measure the anti-HPA-1a activity in patient's samples. Further studies are required to determine the relationship between anti-HPA-1a activity and clinical outcome in patients with neonatal alloimmune thrombocytopenia (NAIT).     

Genetic typing of human platelet antigen 1 (HPA-1) by oligonucleotide ligation assay in a specific and reliable semi-automated system

Genotyping of platelet alloantigens with the possibility of using any type of cellular material as a source of DNA has become a preferred procedure, particularly in thrombocytopenic patients when platelet counts are too low for phenotyping. Recently human platelet antigen 1 (HPA-1) has been identified as an inherited risk factor for coronary thrombosis. The different detection methods currently used have disadvantages for large-scale DNA diagnosis, including the need for electrophoresis (allele-specific restriction enzyme analysis, amplification with sequence-specific primers) or the potential risk of reduced specificity (allele-specific oligonucleotide hybridization). In this report we describe the adaptation of an automated oligonucleotide ligation assay to genotype HPA-1 in polymerase chain reaction (PCR)-amplified DNA samples. HPA-1a and HPA-1b phenotypes corresponded to the results of the different genotyping assays. The genotypes determined with the ELISA-based PCR-oligonucleotide ligation assay were in 100% concordance with the results obtained by conventional allele-specific restriction enzyme site analysis and PCR amplification with sequence-specific primers. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to genotype human platelet antigens that can rapidly be applied to large population screening.     

An International Reference Reagent for the detection of anti‐human neutrophil antigen‐1a

Background and Objectives Testing for neutrophil antibodies has become more common as awareness of transfusion‐related acute lung injury (TRALI) has increased. However, unlike other areas of blood cell antibody testing, there are no certified reference reagents available with which laboratories can determine the sensitivity of detection of their assays. This report describes the production and evaluation of a freeze‐dried preparation of human plasma, code 09/284, containing anti‐human neutrophil antigen‐1a (anti‐HNA‐1a) for use as a minimum sensitivity reagent. Materials and Methods One‐millilitre of aliquots of plasma containing anti‐HNA‐1a were freeze‐dried in glass ampoules. To characterize the material, 24 laboratories took part in an international collaborative study. The participants evaluated doubling dilutions of the material using their in‐house routine assays and recorded the highest dilution in which the antibody could be detected. Results When diluted 1 in 4, most laboratories were able to detect the anti‐HNA‐1a in the material, and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. Conclusions In October 2011, the WHO Expert Committee on Biological Standardization approved the material 09/284 as an International Reference Reagent for the detection of anti‐HNA‐1a.     

Human platelet antigen-1a antibodies induce the release of the chemokine RANTES from human platelets

BACKGROUND AND OBJECTIVE: Binding of human platelet antigen-1a (HPA-1a)-specific antibodies to target platelets can trigger platelet activation and mediator release. Here we tested the effect of HPA-1a antibody-containing sera on platelet release of the chemokine RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) in vitro. PATIENTS AND METHODS: HPA-1a-containing sera obtained from 11 mothers delivered of an infant with neonatal alloimmune thrombocytopenia (NAIT) and from six patients with post-transfusion purpura (PTP) were incubated with HPA-1a/a target platelets. Antibody-induced release of soluble RANTES was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: A significant release of soluble RANTES was induced by four out of the 17 sera. Two out of the four reactive sera were obtained from mothers who were delivered of a baby with NAIT and the remaining two sera were from patients with PTP. Chemokine release was specific for binding of anti-HPA-1a to the platelet membrane, as none of the reactive sera induced the release of soluble RANTES when incubated with HPA-1b/b platelets. The blockade of platelet-expressed Fc gamma receptor type II (FcgammaRII) inhibited anti-HPA-1a-mediated RANTES release when incubated with the reactive sera of patients with NAIT, but not when platelets were incubated with sera of patients with PTP. CONCLUSION: Our findings suggest that anti-HPA-1a antibody-induced release of platelet-derived RANTES can play a role in adverse reactions in alloimmunized patients.     

Interlaboratory variation in the detection of clinically significant alloantibodies against human platelet alloantigens

This report describes the results of eight workshop exercises which were designed to test the proficiency of laboratories in the detection of antibodies to human platelet antigens (HPA). Detection of the most clinically significant alloantibody, anti-HPA-1a, is adequate. However, despite improvements in consistency of test results between laboratories over the last 3 years, there is still a high probability that clinically significant antibodies against other HPA alloantigens will not be detected.     

Severe fetomaternal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-1a in a mother with a rare and silenced ITGB3*0101 (GPIIIa) allele

BACKGROUND AND OBJECTIVES: Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal antibodies against a human platelet antigen (HPA) present on fetal, but absent from maternal platelets. We identified and characterized a case of FMAIT due to anti-HPA-1a in a mother with an HPA-1a1b genotype. MATERIALS AND METHODS: The first child of a 29-year-old mother presented with a petechial rash and a platelet count of 8 x 10(9) per l. Upon routine serological investigation, a discrepancy between the HPA-1a genotype and phenotype prompted the sequencing of the 15 exons of the ITGB3 (integrin beta3, GPIIIa and CD61) gene in the mother. RESULTS: The mother was genotypically HPA-1a1b heterozygous but phenotyped as HPA-1a negative. Sequencing of the ITGB3 exons confirmed HPA-1a1b heterozygosity, but also identified a novel single nucleotide insertion in exon 10 leading to a frameshift and premature termination at amino acid 471 of ITGB3. Maternal anti-HPA-1a was detected but with a pattern typical for a low-affinity antibody. Three transfusions of HPA-1a and -5b negative neonatal platelet concentrates were required to return to a safe platelet count. CONCLUSION: A rare ITGB3 allele was uncovered by the investigation of a severe case of alloimmune thrombocytopenia in a mother with HPA-1a antibodies who genotyped as HPA-1a1b.     

Report on the 15th International Society of Blood Transfusion platelet immunology workshop

Background and Objectives The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet‐reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti‐HPA‐1a and serologically difficult‐to‐assess antibodies against HPA‐3, to identify whether anti‐HPA‐1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. Materials and Methods Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. Results Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA‐1a, in contrast, gave largely congruent results. Serologically difficult‐to‐assess antibodies recognizing HPA‐3a and HPA‐3b were not detected by many laboratories. For genotyping, good agreement was achieved. Conclusions Detection of HPA‐1a antibodies, titration of anti‐HPA‐1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA‐3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable.     

The First International Standard for Insulin-like Growth Factor-1 (IGF-1) for immunoassay: Preparation and calibration in an international collaborative study

The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) has recognized the need for an International Standard for Insulin-like Growth Factor-1 (IGF-1) for the calibration of immunoassays and for the monitoring of the content of therapeutic products. The objective of the study reported here was the characterization of a candidate standard for IGF-1 in an international collaborative study carried out by 18 laboratories in nine countries, by comparison with (i) a primary calibrant characterized by amino acid analysis and UV spectroscopy, and (ii) the existing International Reference Reagent coded 87/518 by HPLC, immunoassay and bioassay. The study was designed as follows: Phase I involved the establishment of a primary calibrant of rhIGF-1, containing approximately 1.0 mg rhIGF-1 per vial. A defined value was assigned to the primary calibrant by amino acid analysis (AAA) and UV spectroscopy. Phase II involved calibration of the candidate standard in terms of the primary calibrant by HPLC, with confirmatory data from immunoassay and bioassay. Results from Phase I confirmed the primary calibrant as containing 1.045 mg per vial. Although there was some variability among laboratory estimates of IGF-1 in the proposed standard using the different methods in Phase II, the estimates by the various methods were in broad agreement. On the basis of the results reported here, the World Health Organization (WHO) has established the preparation coded 02/254 as the First International Standard for Insulin-like Growth Factor-1, human, recombinant, for immunoassay with an assigned content of 8.50 μg per ampoule. Details of how to order the standard can be found at www.nibsc.ac.uk.     

Immunoglobulin G subclasses of anti-human platelet antigen la in maternal sera: relation to the severity of neonatal alloimmune thrombocytopenia

Abstract: The monoclonal antibody immobilization of platelet antigen (MAIPA) technique was employed to detect and semiquantitatively assess total IgG anti-HPA-1a and its subclasses in sera of mothers who gave birth to severely thrombocytopenic (<50×10⁹ platelets/L) (n = 14) or mildly thrombocytopenic/unaffected (>50×10⁹ platelets/L) (n = 13) neonates. There was no statistically significant difference between the IgG anti-HPA-1a subclass composition of the 2 groups of sera. The majority of sera (26/27,96%) showed IgG1+IgG3 while 17/27 (63%) had all 4 subclasses of the antibody. No significant differences between the severely thrombocytopenic and mildly thrombocytopenic/unaffected groups were detected in the levels of IgG, IgG1, IgG2 or IgG4 of the antibody. However, the values of IgG3 anti-HPA-1a were significantly higher in the severely thrombocytopenic than in the mildly thrombocytopenic/unaffected group of sera with only little overlap (median 2.94 vs. 1.68, range 1.36–9.71 vs. 1.50–2.84, respectively; p <0.01). The results suggest that maternal IgG3 anti-HPA-1a has predictive value for severe thrombocytopenia of the neonate. However, a prospective study of IgG HPA-1a subclasses in a greater number of maternal sera at different times of pregnancy is needed to test if IgG3 anti-HPA-1a is predictive of the degree of fetal/neonatal thrombocytopenia.     

Thrombin-inducible platelet adhesion and regulation of the platelet seven-transmembrane thrombin receptor-1 (PAR-1): Effects of unfractionated heparin and lepirudin

Heparin may induce platelet activation and even heparin-induced thrombocytopenia. Lepirudin has been approved for HIT treatment. We speculated that lepirudin inhibits platelet function under high shear and the platelet thrombin receptor PAR-1 better than heparin. Thrombin-inducible platelet adherence under high shear conditions and the expression of PAR-1 were studied after samples from healthy donors were exposed in vitro to increasing concentrations of unfractionated heparin or lepirudin. Compared to baseline and to lepirudin, heparin induced platelet P-selectin expression (p = 0.04). Platelet adherence increased slightly in the presence of lepirudin, but not heparin (p = 0.04). Thrombin-inducible platelet aggregate formation and consecutive adherence under high shear conditions was inhibited by both anticoagulants (p = 0.004). Further, heparin and lepirudin inhibited thrombin-inducible cleavage and internalization of PAR-1 at a dosage of 1.0 U/ml and 1.6 µg/ml, respectively (p = 0.004). Thus, heparin and lepirudin inhibit thrombin-inducible platelet activation in vitro to a similar extent.     

A novel antigen-specific capture assay for the detection of platelet antibodies and HPA-1a phenotyping

BACKGROUND AND OBJECTIVES: The antigen-specific assays currently used for the laboratory investigation of platelet antibodies and antigens are technically complex and cannot be used in most routine laboratories. Here, we describe a simple antigen-specific capture assay (ASCA) for the detection of serum platelet antibodies and for human platelet antigen-1a (HPA-1a) phenotyping. MATERIALS AND METHODS: For the detection of platelet antibodies, platelets from healthy blood donors were incubated with biotinylated monoclonal antibodies to platelet glycoprotein complexes (GP), then solubilized and mixed with superparamagnetic streptavidin particles. Serum samples from patients with autoimmune thrombocytopenia (n = 39), from patients with platelet alloantibodies (6 HPA-1a, 1 HPA-2b, 1 HPA-3a, 6 HPA-5b), and from healthy blood donors (n = 70), were tested. All serum samples from the patients were investigated in parallel by the indirect monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). For HPA-1a phenotyping, superparamagnetic particles were coated with a monoclonal antibody to HPA-1a and mixed with diluted whole blood samples from healthy blood donors (n = 139), who had previously been genotyped for platelet alloantigens. Results The indirect MAIPA detected autoantibodies in 18%, and the direct MAIPA in 50% of patients tested. In contrast, the new ASCA demonstrated positive results in 77% of patients. All tested alloantibodies reacted positive by the ASCA, and all serum samples from healthy blood donors were negative. The results of HPA-1a phenotyping were in concordance with those of genotyping in all cases. CONCLUSION: In our opinion, the ASCA is easy to perform and much more sensitive than the currently available antigen-specific assays for the detection of platelet antibodies.     

Incidence of transfusion-induced platelet-reactive antibodies evaluated by specific assays for the detection of human leucocyte antigen and human platelet antigen antibodies

BACKGROUND AND OBJECTIVES: The aim of this work was to study the incidence of transfusion-induced platelet-reactive antibodies in a selective patient population and evaluate different methodologies for platelet antibody screening (PAS). MATERIALS AND METHODS: The patients were retrospectively selected and divided into three separate groups: haematological malignancies (Group 1: n = 33); cardiac and orthopaedic patients (Group 2: n = 31) and a control group (Group 3: n = 23) selected with the same diagnoses of Group 2. PRE- and POST-transfusion samples were tested for PAS by the following tests: PIFT (platelet immunofluorescence test), MAIPA (monoclonal antibody immobilization of platelet antigen), Flow PRA(R) and LCT (lymphocytotoxicity test). RESULTS: There was not a 100% concordance among the methodologies used. PIFT, MAIPA and Flow PRA presented very similar results whereas that of LCT differed from the other methods. A high rate of positive results (32%) was found in the PRE samples followed by an increase of almost 50% after blood transfusion (POST samples: 42.5% of positivity), but there was a statistical difference (P < 0.05) between the PRE and POST transfusion sample only for the Flow PRA(R) technique tested on Group 2. Human leucocyte antigen (HLA) class I antibodies were present on 97.4% of POST positive samples, 5.4% presented anti-human platelet antigen (HPA)-1b antibodies and 8.1% presented a mix of pan-reactive antibodies against glycoprotein IIbIIIa, IaIIa and IbIX. CONCLUSIONS: Blood transfusion did not increase the rate of alloimmunization in our haematological patients (Group 1); however, the patients were already admitted with a high rate of alloimmunization (12%). Group 2 patients are being immunized and the impact of this procedure remains to be studied as these patients may eventually undergo further hospitalization and receive more blood transfusion.     

Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCyclerTM

Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single-tube method for HPA-1 genotyping that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescently-labelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully complementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCyclerTM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.     

A modified rapid monoclonal antibody-specific immobilization of platelet antigen assay for the detection of human platelet antigen (HPA) antibodies: a multicentre evaluation

BACKGROUND: The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay is the cornerstone technique for the detection and identification of human platelet antigen (HPA) antibodies. However, the original technique described by Kiefel and colleagues requires approximately 8 h adding to diagnostic delay. Moreover, proficiency exercises indicate that there are substantial variations in the MAIPA protocol, and that these may account for interlaboratory differences in sensitivity and specificity. STUDY DESIGN AND METHODS: A review of current MAIPA assay protocols from six laboratories together with performance in quality-assessment schemes identified several key variables potentially affecting the assay results. An optimized protocol was derived and assay time reduced to 5 h. The modified rapid MAIPA (MR-MAIPA) assay was evaluated using 61 samples with a range of HPA antibodies typically encountered in cases of fetomaternal alloimmune thrombocytopenia (n = 22), post-transfusion purpura (n = 8), platelet refractoriness (n = 7) and other platelet immune conditions (n = 24). The sensitivity of the assay was assessed using three international standards and the recombinant HPA-1a antibody CamTran007. The results obtained were compared with the original findings obtained with the local MAIPA assays. In addition, four different glycoprotein IIb/IIIa capture monoclonal antibodies were evaluated for their effect on assay sensitivity. RESULTS: Complete concordance was found between the original MAIPA results and those obtained with the new assay when testing a selected panel of clinical samples. The modified assay had nanogram level sensitivity for the detection of HPA-1a antibodies and titration of HPA-1a and HPA-5b antibody sensitivity standards yielded end-points equal to or greater than the mean recorded in international workshops. CONCLUSION: The MR-MAIPA assay offers improved turnaround for the detection of HPA antibodies without loss of sensitivity.     

An International Standard for specifying the minimum potency of anti-D blood-grouping reagents: evaluation of a candidate preparation in an international collaborative study

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate a lyophilized monoclonal immunoglobulin M (IgM) anti-D preparation for use as an International Standard to specify a recommended minimum acceptable potency of anti-D blood-grouping reagents. MATERIALS AND METHODS: The candidate International Standard (99/836) for specifying the minimum potency of anti-D blood-grouping reagents was evaluated against a wide range of commercial anti-D blood-grouping reagents in an international collaborative study involving 20 laboratories in 13 countries. Laboratories titrated reconstituted 99/836, in parallel with as many commercial anti-D blood-grouping reagents as were available to them, in tube tests according to specified haemagglutination methodology for low-protein (e.g. monoclonal IgM) and high-protein (e.g. polyclonal) reagents. The ratios of the mean end-point titres of the reagents to that of 99/836 within each laboratory were calculated. RESULTS: The ratios of the mean titres of the low-protein reagents to the mean titre of 99/836 within a laboratory fell between 0.25 and 2 for 43 of the 45 low-protein anti-D reagents tested (i.e. the potencies of the low-protein reagents compared with 99/836 were between a 1:4 dilution of 99/836 to twice as potent as 99/836). The ratios of the mean titres of the high-protein reagents to the mean titre of 99/836 within a laboratory fell within 0.125 and 1 for eight out of the 10 high protein reagents tested. CONCLUSIONS: By international consensus, a 1:3 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of low-protein anti-D blood-grouping reagents. A 1:8 dilution of reconstituted 99/836 was deemed appropriate to define a recommended minimum acceptable potency of high-protein anti-D blood-grouping reagents. On the basis of the results presented here, 99/836 was established by the World Health Organization as the 1st International Standard for specifying the minimum potency of anti-D blood-grouping reagents, in tube tests.     

The exosome of platelet endothelial cell adhesion molecule-1 (PECAM1) protein: A potential risking star in high blood pressure patients (HBPP)

A number of studies have demonstrated that exosomes were involved in important physiological and pathological processes through cell-to-cell communication in cardiovascular disease, which contained nucleic acids, proteins, and lipid contents. In our study, we found that the protein platelet endothelial cell adhesion molecule-1 (PECAM1) was an extracellular vesicle in the blood of high blood pressure patients (HBPP).Isolated the vesicles from the blood of HBPP and health examiners and detected its size and morphology with nanoparticle tracking analysis, then we identified its surface protein CD63, CD81, and the protein expression of PECAM1 in the exosome with western blot. Furthermore, we analyzed the correlation between the expression of PECAM1 and the high blood degree with linear regression analysis.Our results showed that the morphology of extracellular vesicles was more evident in high blood pressure groups than healthy controls, and the protein expression of PECAM1 was also abundant in the vesicles of HBPP, however, there were no extracellular vesicles in the blood samples of healthy controls. Besides, linear regression showed the linear correlation coefficient R = 0.901, P < .01 between the expression of PECAM1 and the systolic blood pressure of the high blood patients. Therefore, the exosome of protein of PECAM1 was a potential risking star in HBPP.