High-Throughput CD36 Phenotyping on Human Platelets Based on Sandwich ELISA and Mutant Gene Analysis.

Background: CD36 deficiency is closely associated with fetal/neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness, and other hemorrhage disorders, particularly in Asian and African populations. There is a clinical need for rapid and high-throughput methods of platelet CD36 (pCD36) phenotyping to improve the availability of CD36 typing of donors and assist clinical blood transfusions for patients with anti-CD36 antibodies. Such methods can also support the establishment of databases of pCD36-negative phenotypes. Study Design and Methods: A sandwich enzyme-linked immunosorbent assay (ELISA) for CD36 phenotyping of human platelets was developed using anti-CD36 monoclonal antibodies. The reliability of the assay was evaluated by calculating the intra-assay and inter-assay coefficients of variation (CV). A total of 1,691 anticoagulant whole blood samples from healthy blood donors were randomly selected. PCD36 expression was measured using a sandwich ELISA. PCD36 deficiency was confirmed by flow cytometry (FC). Mutations underlying pCD36 deficiency were identified using polymerase chain reaction sequence-based typing (PCR-SBT). Results: The sandwich ELISA for pCD36 phenotyping had high reliability (intra-assay CV, 2.1-4.8%; inter-assay CV, 2.3-5.2%). The sandwich ELISA was used to screen for CD36 expression on platelets isolated from 1,691 healthy blood donors. Of these, 36 samples were pCD36-negative. FC demonstrated absence of CD36 expression on monocytes in three of the 36 cases. In the present study population, the frequency of CD36 deficiency was 2.13% (36/1,691), of which 0.18% (3/1,691) was type I deficiency and 1.95% (33/1,691) was type II deficiency. In addition, we used PCR-SBT to characterize the gene mutations in exons 3-14 of the CD36 gene in 27 cases of CD36 deficiency and discovered 10 types of mutations in 13 pCD36-negative samples. Conclusion: The present study describes the development and characterization of a highly reliable sandwich ELISA for high-throughput screening for pCD36 expression. This novel method is feasible for clinical applications and provides a useful tool for the establishment of databases of pCD36-negative phenotype donors.

A novel enzyme-linked immunostaining technique based on silk membrane for the prenatal detection of fetomaternal haemorrhage.

Objective: Developing a simple, rapid, reliable, sensitive, and cost-effective method for prenatal detection of fetomaternal haemorrhage by combining multi-aperture silk membrane with enzyme-linked immunosorbent assay (ELISA), which does not require any complicated instruments and can be visually colored, so as to provide a new method for clinical detection of fetomaternal haemorrhage. Methods: As a carrier, a chemically treated silk membrane was used to immobilize anti-A/anti-B antibody reagent. PBS washed slowly after vertically dropping red blood cells. After adding biotin-labeled anti-A/anti-B antibody reagent, PBS is slowly washed, enzyme-labeled avidin is added, and TMB is used for color development after washing. Results: When there were both anti-A and anti-B fetal erythrocytes in pregnant women's peripheral blood, the final color was dark brown. When there are no anti-A and anti-B fetal red blood cells in pregnant women's peripheral blood, the final color development results do not change, which corresponds to the color of chemically treated silk membrane. Conclusion: The new enzyme-linked immunosorbent assay (ELISA) based on a silk membrane can distinguish fetal red blood cells from maternal red blood cells prenatally and can be used for prenatal detection of fetomaternal haemorrhage.

Prenatal diagnosis of fetomaternal hemorrhage by a novel hydrogel fluoroimmunoassay that accurately quantifies fetal haemoglobin.

Objective: Fetomaternal hemorrhage (FMH) is an alloimmunization resulting caused by the incompatibility between fetal and maternal blood. For the prevention of newborn haemolytic disease (HDN), it is crucial to quantify the amount of fetomaternal hemorrhage. However, the classical Kleihauer-Betke test (K-B test) for detecting fetomaternal hemorrhage is limited by experimental tools and conditions and is not suitable for routine clinical use. Consequently, the method of prenatal diagnosis of fetomaternal hemorrhage applicable to the clinic is a topic worthy of further study. Therefore, it is worthwhile to further investigation on the clinically applicable prenatal diagnosis method for fetomaternal hemorrhage. Methods: This experiment demonstrates hydrogel's ability to separate sensitized red blood cells from soluble antibodies. Using flow cytometry the fluorescence values of sensitized red blood cells and fluorophore-labeled antibodies were measured, and the testing steps for the detection products of a novel technology were determined. The properties of a hydrogel fluoroimmunoassay were evaluated by distinguishing between the amounts of fetal and adult haemoglobin. The precision of this technology is evaluated using the Kleihauer-Betke test as a comparison. Results: This experiment compared the detection of haemoglobin fluorescence in adults (n = 2) and fetuses (n = 6). At the same time, the fluorescence intensity of different fetal haemoglobin (HbF) in adult haemoglobin (HbA) was calculated. The fluorescence value is 1.6% when the fetal hemoglobin concentration is 0.1%. Conclusion: The novel hydrogel fluoroimmunoassay can accurately determine the fluorescence intensity by flow cytometry to differentiate fetal haemoglobin from adult haemoglobin, quantitatively prenatally diagnose fetal haemoglobin, address the incompatibility between fetal and maternal blood types, and prevent alloimmunization.

The development and application of a novel reagent for fixing red blood cells with glutaraldehyde and paraformaldehyde.

OBJECTIVE: The currently employed red blood cell reagents have a short shelf life. Some hospitals with a small number of specimens will be unable to utilize them within the validity period, resulting in a substantial increase in the purchase price. Therefore, the method of developing long-term red blood cell reagents is a problem worthy of further study. METHODS: In this experiment, the type and concentration of the red blood cell reagent treatment solution were evaluated based on the red blood cell antigen concentration 24 h after treatment. In addition, the qualified glutaraldehyde/paraformaldehyde reagent was stored for six months, and five red blood cell indices were measured every month. At the same time, the detection indices of treated red blood cell reagents and untreated red blood cell reagents were compared. RESULTS: It was discovered that treated red blood cells containing 0.005% GA and 0.05% PFA were more suitable for the preservation of red blood cells than other treated concentrations, and the preservation time could reach six months. The test tube method (n = 24) and microcolumn gel card (n = 35) were used to determine the accuracy of the treated blood cells containing 0.005% glutaraldehyde +0.05% paraformaldehyde, with an accuracy of 100%. CONCLUSION: This experiment resulted in the development of a novel reagent for treating red blood cells with glutaraldehyde/paraformaldehyde fixed solution that can effectively prolong its storage time by two to three times that of red blood cell reagents currently on the market.

Centrifugal microfluidic platform with digital image analysis for parallel red cell antigen typing.

This study presents a portable multichannel microfluidic device for parallel and digital analysis of red cell antigen typing. A zigzag-shaped precise metering channel was designed for the simultaneous aliquoting of samples, which is independent of the volume of the predeposited blood-typing reagents in the reaction chambers. The entire assay protocol can be conducted using a sequential-step spinning protocol, which resembles that of conventional tube tests for blood typing; however, the manual procedure is largely reduced compared to that of conventional systems. After loading the samples, the disc is centrifuged in a defined program with five sequential steps, each of which can be completed in a few seconds. Through step-wise centrifugation, predeposited antibodies react with red blood cells, enabling the parallel identification of multiple red blood cell antigens without cross-contamination in 1 min. This is combined with gentle mixing to rapidly concentrate the agglutinates, making both visual and digital determination of agglutination straightforward. A customized image analysis algorithm for automatically determining the agglutination state was developed to complement this microfluidic system. The acquired image is processed after the test. The blood type is determined using a machine learning algorithm based on a histogram of oriented gradients (HOG) and support vector machines (SVM). This allows digital analysis to mirror the classical laboratory procedure for blood-type determination more accurately. The system was trained using a validated dataset of 150 blood samples, presenting 750 different agglutination patterns. The combination of SVM and HOG achieved 94.10% in the micro-weighted performance evaluation. This integrated microfluidic chip-based platform provides a "sample-in and answer out" demonstration for red blood cell typing, ensuring fast and reliable results because minimum manual steps are involved.

Study on a Natural Silk Cocoon Membrane-Based Versatile and Stable Immunosensing Platform via Directional Immunoaffinity Recognition.

The development of immunosensing assays for in vitro diagnostics has attracted great attention in recent years. Various substrate materials and immobilization methods of biomolecules were exploited for immunosensors, but their bioactivity and longevity have been facing serious challenges. To address this limitation, we investigated a natural silk cocoon membrane as immunosensing substrate material. By using its intrinsic properties, the target biomolecules were immobilized on the membrane through directional immunoaffinity recognition. The silk cocoon membrane-based immunosensor showed great potential for both qualitative and quantitative immunoassays, through naked-eye observation or analyzing the change in red color intensity, respectively. The immunosensor exhibited significant detection capability for anti-D (titer 1:1024) sensitized red blood cells. The colorimetric responses of concentrations ranged from 1 µg/mL to 1 ng/mL, and the detection limit for anti-D was 3.4 ng/mL. The immunosensor also showed excellent stability for the immobilized antibodies when stored at 4 and 25 °C; the bioactivity remained unchanged or slightly declined within 40 weeks. Even at 37 °C, the bioactivity began to decline after 12 weeks. This current work highlights the potential of using the natural silk cocoon membrane as a substrate for a versatile and thermally stable immunosensing platform for application in immunoassays.

Dual-band fluorescence detection of double-stranded DNA with QDs-Mn2+-pefloxacin.

By integrating the fluorescence of quantum dots (QDs) and Mn2+-pefloxacin mesylate (Mn2+-pefloxacin), a new type of dual-band fluorescence biosensor for high-efficiency and sensitive determination of double-stranded DNA (dsDNA) is developed. The biosensor is based on the fluorescence "OFF-ON" mode of both QDs and QDs-Mn2+-pefloxacin. The Mn2+-pefloxacin complex can quench the QDs fluorescence via photoinduced electron transfer (PET), and its fluorescence is also quenched. Due to the specificity and strong binding affinity of dsDNA for the Mn2+-pefloxacin complex, it can break the low fluorescent QDs-Mn2+-pefloxacin and restore the fluorescence of QDs and Mn2+-pefloxacin complex in their respective bands. Therefore, the dual-band fluorescence quantitative detection of dsDNA by QDs-Mn2+-pefloxacin can be achieved, while bovine serum albumin, single-stranded DNA, and bio-related ions do not yield similar results. Furthermore, the possible reaction mechanisms are systematically discussed. The detection limits (3δ/K) of herring sperm (hs) DNA in the fluorescence recovery bands of QDs and Mn2+-pefloxacin complex are 0.0142 and 0.0465 µg/mL, respectively. The developed biosensor was used for dsDNA detection in synthetic samples, and desirable results are obtained.

A CNN-LASSO ensemble classification model for incomplete antibody reactants screening in coombs test.

BACKGROUND: Precise classification of incomplete antibody reactants (IAR) in the Coombs test is the primary means to prevent incompatible blood transfusions. Currently, an automatic and contactless method is required for accurate IAR classification to avoid human error. OBJECTIVE: We present an ensemble learning algorithm that integrates five convolutional neural networks and the least absolute shrinkage and selection operator (LASSO) regression algorithm into an IAR intensity classification model. METHODS: A dataset including 1628 IAR and corresponding labels of IAR intensity categories ((-), (1+), (2+), (3+), and (4+)) was used. We trained the ensemble model using 1302 IAR and validated its performance using 326 IAR. The optimal ensemble model was used to assist immunologists in classifying IAR. The chord diagrams based on the human-machine interaction were established. RESULTS: The ensemble model achieved 98.8%, 98.4%, 99.7%, 99.5%, and 99.4% accuracies in the (-), (1+), (2+), (3+), and (4+) categories, respectively. The results were compared with those of manual classification by immunologists (average accuracy: 99.2% vs. 75.6%). Using the model, all three immunologists achieved increased accuracy (average accuracy: +8.4%). CONCLUSIONS: The proposed algorithm can thus effectively improve the accuracy and efficiency of IAR intensity classification and facilitate the automation of haemolytic disease screening equipment.

Convolutional neural network-based automatic classification for incomplete antibody reaction intensity in solid phase anti-human globulin test image.

The precise classification of incomplete antibody reaction intensity (IARI) in hydrogel chromatography medium high density medium solid-phase Coombs test is essential for haemolytic disease screening. However, an automatic and contactless method is required for accurate classification of IARI. Here, we present a deep ensemble learning model that integrates five different convolutional neural networks into a single model for IARI classification. A dataset, including 1628 IARI images and corresponding labels of IARI categories ((-), (1 +), (2 +), (3 +), and (4 +)), was used. We trained our model using 1302 IARIs and validated its performance using 326 IARIs. The proposed model achieved 100%, 99.4%, 99.4%, 100%, and 100% accuracies in the ( -), (1 +), (2 +), (3 +), and (4 +) categories, respectively. The results were compared with those of manual classification by immunologists (average accuracy: 99.8% vs. 88.3%, p < 0.01). Following model assistance, all three immunologists achieved increased accuracy (average accuracy: + 6.1%), with the average accuracy of junior immunologists maximum increasing by 11.3%. The time required for model classification was 0.094 s·image-1, whereas that required manually was 5.528 s·image-1. The proposed model can thus substantially improve the accuracy and efficiency of IARI classification and facilitate the automation of haemolytic disease screening equipment.

Study on the antigens and antibodies of Mur and Mia blood groups in southern China.

BACKGROUND: The frequency of Mur and Mia blood group antigens in Asian population is much higher than that in Caucasian population. However, due to the scarcity and high price of commercial detection reagents, there are few studies on antigen and antibody detection and comparative analysis in large samples. OBJECTIVE: To study the occurrence frequency, antigen correlation and antibody properties of Mur and Mia antigens and their corresponding antibodies in southern China. METHODS: Mur and Mia antigens and antibodies in local blood donors and patients were detected by routine serological microplate method. Statistical methods were used to calculate the incidence of two antigens and antibodies and analyze their correlation. RESULTS: Among blood donors, the positive rates of Mur and Mia antigens were 6.4 % and 6.5 % respectively, with no significant difference (P > 0.05). In this region, the incidence of anti-"Mur" and anti-"Mia" was 0.65 % and 0.45 % respectively. But significant difference existed between blood donors and patients (P < 0.05). Among the anti-"Mur" and anti-"Mia" positive patients, most of the antibodies were IgM or IgM + IgG mixed type and had saline activity. CONCLUSION: Mur and Mia antigens and their corresponding antibodies have a high frequency in the population of southern China. In the routine clinical detection of irregular antibodies, Mur or Mia positive (GP.Mur) should be added to screen erythrocytes. Moreover, given the high correlation between Mur and Mia antigens expression on red blood cells, using monoclonal antibodies against Mia could predict the presence of Mur antigen.

Chicken Egg Yolk Antibodies (IgYs) block the binding of multiple SARS-CoV-2 spike protein variants to human ACE2.

The SARS-CoV-2 virus is still spreading worldwide, and there is an urgent need to effectively prevent and control this pandemic. This study evaluated the potential efficacy of Egg Yolk Antibodies (IgY) as a neutralizing agent against the SARS-CoV-2. We investigated the neutralizing effect of anti-spike-S1 IgYs on the SARS-CoV-2 pseudovirus, as well as its inhibitory effect on the binding of the coronavirus spike protein mutants to human ACE2. Our results show that the anti-Spike-S1 IgYs showed significant neutralizing potency against SARS-CoV-2 pseudovirus, various spike protein mutants, and even SARS-CoV in vitro. It might be a feasible tool for the prevention and control of ongoing COVID-19.

Application of lyophilised human platelets for antibody detection in solid phase red cell adherence assay.

Antibodies against human platelets cause a variety of thrombocytopenic disorders, which lead to potentially fatal haemorrhage. Therefore, their prompt detection is mandatory for successful patient treatment. Solid phase red cell adherence (SPRCA) assay allows for platelet antibody detection widely. However, preparation of fresh platelets with HLA-I and human platelet antigens (HPA)1-5,15 genotyped as target cells is inconvenient and fresh platelets have a short shelf life. In this study, the lyophilised human platelets for antibody detection in SPRCA were prepared. Firstly, platelets were resuspended in lyophilisation buffer and freeze-dried. Then the characteristics of lyophilised platelet were analysed. Rehydrated platelets were recovered with a mean rate of 80.91% ± 2.87%, and still retained spherical morphology. Indirect flow cytometry showed that glycoproteins IIb/IIIa, Ia/IIa, Ib/IX, IV, CD109, and HLA class I were present on the surface of the lyophilised platelets at a comparable level to that of fresh platelets. The consistent results obtained with WHO reference reagents containing anti-HPA-1a, anti-HPA-3a, and anti-HPA-5b, as well as clinical samples from the same donors containing anti-HLA antibodies when reacting with lyophilised versus fresh platelets confirmed good antigenicity preservation of platelets after freeze-drying. Further investigation showed that the lyophilised platelets could be stored at 2-8 °C for up to 14 months and the reconstituted suspension was stable for 48 h. Therefore, lyophilised platelets can be a convenient alternative to fresh platelets to use for anti-platelet antibody detection in SPRCA tests.

An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products.

Real-time polymerase chain reaction (RT-PCR) enables effective and sensitive screening for infectious risk in the field of blood safety. However, when using RT-PCR to detect bacterial contamination, several intractable points must be considered, one of which is the lack of appropriate quality control. In this study, we developed a simplified RT-PCR assay in which the same primer set and two distinct probes were used to detect both, an internal reference control and the target in a reaction. The copy number of the internal reference control represents the positive detection limit of the assay; therefore, when the threshold-cycle value of the target is less than or equal to that of the internal reference control, the result obtained for the target can be considered to be a true positive. When human gDNA was spiked with Escherichia coli gDNA and the detection limit for the internal reference control was set to five copies, the measured detection limit for E. coli gDNA was two copies. The internal reference control duplex RT-PCR assay showed high efficiency (0.91-1.02), high linearity (R2 > 0.99), and good reproducibility in intra- and inter-assay comparisons. Lastly, when human platelet-rich plasma samples were spiked with E. coli or other bacterial species, all species were detected efficiently, and the results of a two-sample pooled t test showed that the limit of detection for E. coli was 1 cfu/mL. Here, we present a synthetic internal reference control molecule and a new statistical method for improving the reliability of RT-PCR assays when screening for bacterial contamination in blood products.

A new approach to detection of incomplete antibodies using hydrogel chromatography medium.

In assays for incomplete antibody detection, several washing steps are required to remove unbound globulins which may cause false negatives. Here, we present an improved approach employing hydrogel chromatography medium (HCM) in the detection of incomplete antibodies. After a rapid single-step centrifugation, incomplete antibodies, attached to red blood cells (RBCs), were separated from the reaction mixture using HCM and sedimentation. This method obviates the need for multiple centrifugation steps found in conventional Tube-Coombs tests. The HCM-Coombs tests may have a wide range of applications for incomplete antibody detection.