Identification Of Antibodies Research Articles

The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence

Moesin is a cytoskeletal adaptor protein, involved in the modification of the actin cytoskeleton, with relevance to Alzheimer’s Disease. Well characterized anti-Moesin antibodies would benefit the scientific community. In this study, we characterized ten commercial antibodies for Moesin in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Red blood cell alloantibodies in paediatric transfusion in sub-Saharan Africa: A new cohort and literature review.

Blood transfusion support predisposes transfused children to the risk of erythrocyte alloimmunization in Sub-Saharan Africa. A cohort of 100 children receiving one to five blood transfusions were recruited for screening and identification of irregular antibodies using gel filtration technique. The mean age was 8 years and the sex-ratio at 1.2. The retrieved pathologies were: major sickle cell anaemia (46%), severe malaria (20%), haemolytic anaemia (4%), severe acute malnutrition (6%), acute gastroenteritis (5%), chronic infectious syndrome (12%) and congenital heart disease (7%). The children presented with haemoglobin levels≤6g/dl, and 16% of them presented positive irregular antibodies directed against the Rhesus (30.76%) and Kell (69.24%) blood group systems. A literature review shows that irregular antibody screenings vary from 17% to 30% of transfused paediatric patients in Sub-Saharan Africa. These alloantibodies are in particular directed against the Rhesus, Kell, Duffy, Kidd and MNS blood group and generally found in sickle cell disease and malaria. This study highlights the urgent need of extended red blood cell phenotyping including typing for C/c, E/e, K/k, and Fya/Fyb, and if possible Jka/Jkb, M/N, and S/s for children before transfusion in Sub-Saharan Africa.

Preparation and Identification of a Monoclonal Antibody against the Pseudorabies Virus gE Glycoprotein through a Novel Strategy.

Since 2011, pseudorabies virus (PRV) has recurred in several vaccinated pig farms in China. PRV variants with high virulence were found to be the main cause of the outbreaks. In the face of the PRV epidemic, detection of the wild strain is as important as vaccine immunization, so we hoped to achieve differential diagnosis of PRV by obtaining a monoclonal antibody (mAB) that could be used to identify the wild strain. In this study, we used a novel immunization and screening strategy to prepare an mAB and obtained mAB 1H5 against the gE glycoprotein. An immunofluorescence assay (IFA) revealed that this mAB was specific to both classic and variant strains of PRV. Subsequently, we further identified the linear epitopes of B cells recognized using the mAB. The mAB 1H5 bound at 67RRAG70, which is a novel epitope and is conserved in almost all PRV strains. These findings provide novel insight into the structure and function of PRV proteins, the analysis of antigenic epitope characteristics, and the establishment of antigen or antibody detection methods.

A novel high-prevalence antigen in the Lutheran system, LUGA (LU24), and an updated, full-length 3D BCAM model.

The basal cell adhesion molecule (BCAM) carries the antigens of the Lutheran (LU, ISBT005) system. We report a novel Lutheran antigen and propose an updated, full-length 3D model of BCAM. Red blood cell testing, antibody identification, and BCAM genomic DNA sequencing were done by standard methods. Multi-template homology modeling of BCAM used structural templates selected for coverage, highest sequence identity, and protein domain family. All variants causing the loss or gain of a Lutheran antigen were analyzed for residue accessibility and intraprotein interactions. An antibody to a high-prevalence antigen in the plasma of a pregnant woman was determined to be directed at a novel Lutheran antigen. Sequencing of BCAM found three homozygous changes: c.212G > A (p.Arg71His) and two silent, c.711C > T and c.714C > T. The model was built from the first two immunoglobulin crystallized domains of BCAM (D1, D2), three other templates (for D3, D4 and D5 with a higher sequence identity with the target than those used for the model proposed by Burton and Brady in 2008, and for the transmembrane region) and RaptorX (for the intracellular domain). All residues associated with a Lutheran antigen were found to be exposed in wild-type or variant proteins, except p.447 associated with loss of Lu13 expression. The c.212G > A change results in the loss of LUGA (LU24) antigen. Whole genome sequencing continues to reveal polymorphisms with uncertain immunogenicity. This model and demonstration that nearly all residues associated with the expression of a Lutheran antigen are exposed will help evaluate the significance of new polymorphisms.

Autoantibodies mimicking alloantibodies: A case series unveiling the dilemmas of transfusion.

Autoimmune hemolytic anemia is characterized by increased red cell destruction and/or decreased red cell survival due to autoantibodies directed against self-antigens on red cells. Since autoantibodies react with self and nonself red blood cells (RBCs), they tend to mask the underlying clinically significant alloantibodies and many a times mimic a specific pattern like alloantibodies. We discuss three immune hematological cases of warm autoantibodies. Antibody screening was performed by solid-phase red cell adherence (SPRCA) technique on a fully automated platform NEO Iris (Immucor Inc., USA). In case of a positive antibody screen, antibody identification was performed using SPRCA, NEO Iris (Immucor Inc., USA). Alloadsorption for adsorbing the autoantibodies was done using in-house prepared allogenic packed RBCs - R1R1, R2R2, and rr. All cases had warm autoantibody with a broad specificity against self-Rh antigens. Anti "C" and Anti "e" antibodies were identified in case 1 and autoanti "e" antibody in cases 2 and 3. Case 3 had underlying alloanti "E" along with autoanti "e" which posed a transfusion challenge. Our case series highlights the importance of detecting the nature of the antibody whether it is alloantibody or autoantibody with antigen specificity. This would help in selecting appropriate antigen negative blood units for transfusion purpose.

Delayed Blood Transfusion Adverse Reactions Caused by Hypothermia Cardiopulmonary Bypass and Its Coping Strategies

To investigate a case of delayed hemolytic transfusion reaction (DHTR), and find the reason and coping strategies to prevent similar incidents in the future. Relative antibody identification was carried out, suitable erythrocytes was screened, and relevant indicators after transfusion were evaluated. Medical record and laboratory report of the patient were reviewed, the cause was analyzed, and corresponding treatment was carried out. The patient suffered from DHTR caused by IgM anti-M antibody and low body temperature cardiopulmonary bypass. After anti-hemolytic treatment, the patient was gradually improved, the bilirubin gradually decreased, and finally cured and discharged. When the transfusion department finds any antibodies which have activity at room temperature but no respond at 37 ℃, they should communicate with clinical medical staff in time. Warm transfusion should be used during blood transfusion to prevent transfusion-related hemolytic reaction.

The RESP AI model accelerates the identification of tight-binding antibodies

High-affinity antibodies are often identified through directed evolution, which may require many iterations of mutagenesis and selection to find an optimal candidate. Deep learning techniques hold the potential to accelerate this process but the existing methods cannot provide the confidence interval or uncertainty needed to assess the reliability of the predictions. Here we present a pipeline called RESP for efficient identification of high affinity antibodies. We develop a learned representation trained on over 3 million human B-cell receptor sequences to encode antibody sequences. We then develop a variational Bayesian neural network to perform ordinal regression on a set of the directed evolution sequences binned by off-rate and quantify their likelihood to be tight binders against an antigen. Importantly, this model can assess sequences not present in the directed evolution library and thus greatly expand the search space to uncover the best sequences for experimental evaluation. We demonstrate the power of this pipeline by achieving a 17-fold improvement in the KD of the PD-L1 antibody Atezolizumab and this success illustrates the potential of RESP in facilitating general antibody development.

Yeast biopanning against site-specific phosphorylations in tau.

The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer's Disease and other neurodegenerative diseases. However, there is a lack of phospho-specific monoclonal antibodies and limited validation of their binding specificity. Here, we report a novel approach using yeast biopanning against synthetic peptides containing site-specific phosphorylations. Using yeast cells displaying a previously validated phospho-tau (p-tau) single-chain variable region fragment (scFv), we show selective yeast cell binding based on single amino acid phosphorylation on the antigen. We identify conditions that allow phospho-specific biopanning using scFvs with a wide range of affinities (KD = 0.2 to 60 nM). Finally, we demonstrate the capability of screening large libraries by performing biopanning in 6-well plates. These results show that biopanning can effectively select yeast cells based on phospho-site specific antibody binding, opening doors for the facile identification of high-quality monoclonal antibodies.

High rates of antibodies against Toscana and Sicilian phleboviruses in common quail Coturnix coturnix birds.

Birds are involved natural cycle of a number of vector-borne viruses in both rural and urban areas. Toscana (TOSV) and Sicilian (SFSV) phleboviruses are sandfly-borne viruses in the genus Phlebovirus that can cause diseases in human. However, there is limited information on the role of the birds in sandfly-borne phleboviruses natural cycle and reservoirs ofthese viruses remain unknown. In this study, we analyzed Common Quail (Coturnix coturnix) sera from Spain to identify the seroprevalence of these two phleboviruses. We tested respectively, 106 and 110 quail serum against TOSV and SFSV from 2018, 2019, and 2021 from two locations in northern Spain with using virus neutralization test. We identified high neutralizing antibody rates for SFSV (45.45%) and TOSV (42.45%) with yearly fluctuation. This is the first identification of SFSV and TOSV neutralizing antibodies in wild birds. High seroprevalence rates of TOSV and SFSV in quail birds raises the question whether birds have a role as amplifying hosts in the natural cycle of phleboviruses.

The rapid and highly parallel identification of antibodies with defined biological activities by SLISY

The therapeutic applications of antibodies are manifold and the emergence of SARS-CoV-2 provides a cogent example of the value of rapidly identifying biologically active antibodies. We describe an approach called SLISY (Sequencing-Linked ImmunoSorbent assaY) that in a single experiment can assess the binding specificity of millions of clones, be applied to any screen that links DNA sequence to a potential binding moiety, and requires only a single round of biopanning. We demonstrate this approach using an scFv library applied to cellular and protein targets to identify specific or broadly reacting antibodies. For a cellular target, we use paired HLA knockout cell lines to identify a panel of antibodies specific to HLA-A3. For a protein target, SLISY identifies 1279 clones that bound to the Receptor Binding Domain of the SARS-CoV-2 spike protein, with >40% of tested clones also neutralizing its interaction with ACE2 in in vitro assays. Using a multi-comparison SLISY against the Beta, Gamma, and Delta variants, we recovered clones that exhibited broad-spectrum neutralizing potential in vitro. By evaluating millions of scFvs simultaneously against multiple targets, SLISY allows the rapid identification of candidate scFvs with defined binding profiles facilitating the identification of antibodies with the desired biological activity.

Significance of Detecting Antierythrocyte Antibodies in Pretransfusion Testing.

Transfusion treatment during life, as well as pregnancy in women, can stimulate the sensitization of the person who received blood, which after the transfusion of blood products can result in the occurrence of moderate to very severe posttransfusion reactions. The aim of this study was to examine the specificity and frequency of antierythrocyte antibodies during the pretransfusion treatment of patients depending on the gender, to determine the origin of antibodies in patients serum, as well as to examine their clinical significance. Retrospective analysis of documentation was performed in the Department for Pretransfusion Testing, therapy and distribution of blood products, Polyclinic for Transfusion, UKC Tuzla. Data was analyzed by reviewing the written and electronic documentation from the period of 5 years (2018-2022). A retrospective analysis of 378 procedures for the identification of antierythrocyte antibodies was performed. It was evident that 140 of all detected antibodies belonged to the Rh-system (66.7%), of which 32.4% were anti RhD-antibodies, 20% anti Rh-E, anti Rh-c 7.1%, and in a low percent of anti-C, anti-e and anti-Cw antibodies. Combinations of anti-D and anti-C antibodies showed the highest frequency (34.2%), followed by a combination of anti-E and anti-c antibodies (21%). Pretransfusion testing represents a very important link in the safety of the use of blood. The identification of antierythrocyte antibodies and the use of phenotyped blood products significantly reduces the risk of posttransfusion reactions and facilitates the implementation of the safe blood policy.

Comparative Study of Tube and Gel Techniques for Compatibility Testing and Antibody Identification

Comparative Study of Tube and Gel Techniques for Compatibility Testing and Antibody Identification

Is Indirect Coombs Test by Pooled Cells Sufficient for Antibody screening? An Eye-Opening Case Series

The ideal way to screen for the presence of alloantibodies is by antibody screening panels which represent all clinically significant antigens in appropriate dosage. Most centers use pooled O-cells for antibody screening as it has antigens of non-ABO blood group systems that are prevalent in a representative population. Pooled O-cells sometimes fail to detect antibodies to less prevalent red blood cell antigens with reduced expression or show a dosage phenomenon. Despite pooling 4 to 5, O-donor segments, sometimes, it is difficult to detect clinically significant antibodies. False-negative indirect Coombs test by pooled O-cells may delay getting a compatible unit for elective cases where type and screen policy is used. Donor units with weak antigenic expression or units showing dosage can come compatible despite being antigen positive and lead to a hemolytic reaction. We report two cases where antibody screening by pooled O-cells was negative; still, cross-match was incompatible. Antibody screening with a three-cell panel was positive. Antibody identification with 11-cell panels confirmed the alloantibody to be anti-E. The present cases emphasize the importance of three-cell panels over pooled O-cells.

Type and Screen Method versus Antihuman Globulin Crossmatch in Pretransfusion Testing: A Cross-sectional Study

Introduction: Blood transfusion remains the primary modality of treatment for many serious and common diseases. According to the International Society of Blood Transfusion (ISBT), there are about 349 blood group antigens, out of which only about 25-28 antigens are known to cause acute or delayed type of haemolytic transfusion reactions which could be prevented by Pretransfusion Testing (PTT). Regulated pretransfusion tests include ABO blood grouping, Rh typing, antibody detection, antibody identification and compatibility testing. The purpose of compatibility tests is to demonstrate invitro red cell antigen-antibody reaction. The Antihuman Globulin (AHG) crossmatch testing can assure ABO compatibility between donor and patient blood as well as detect most clinically significant antibodies. Type and Screen (T&S) is a procedure carried out as part of PTT in which the recipient’s blood sample is tested for ABO group, RhD T&S for unexpected antibodies. Aim: To compare T&S method of PTT with AHG crossmatch. Materials and Methods: This cross-sectional study was conducted in the department of Transfusion medicine at Government Royapettah Hospital, Chennai and The Tamil Nadu Dr. MGR Medical University, Chennai, Tamil Nadu, India from June 2012 to December 2013. T&S was performed on 1,040 recipients’ (510 males and 530 females) samples. All these samples were subjected to AHG crossmatch with ABO group and RhD type matched donor samples to assess the compatibility between donor and recipient by using column agglutination technology. Statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS) version 11.0. Results: The prevalence of unexpected antibodies in the recipient population was 1.06%. Among the 1,040 recipients’ blood samples, 11 samples were found to have unexpected antibodies. Out of these 11 samples, 10 showed exact antibodies and the remaining one sample with negative antibody screening was found to be incompatible with AHG crossmatch. The sensitivity and specificity of T&S method in comparison to AHG crossmatch was 87.50% and 99.71%, respectively. Conclusion: The sensitivity and specificity of T&S is as acceptable as AHG crossmatch. However, in view of one sample with false negative antibody screening in the study population, it is imperative to know the phenotyping of Red Blood Cells (RBC) antigens of the native population before getting away with AHG crossmatch.

Identification of high-performing antibodies for SPARC-related modular calcium-binding protein 1 (SMOC-1) for use in Western Blot and immunoprecipitation.

SPARC-related modular calcium-binding protein 1, otherwise known as SMOC-1, is a secreted glycoprotein involved in various cell biological processes including cell-matrix interactions, osteoblast differentiation, embryonic development, and homeostasis. SMOC-1 was found to be elevated in asymptomatic Alzheimer's disease (AD) patient cortex as well as being enriched in amyloid plaques and in AD patientcerebrospinal fluid, arguing for SMOC-1 as a promising biomarker for AD. Having access to high-quality SMOC-1 antibodies is crucial for the scientific community. It can ensure the consistency and reliability of SMOC-1 research, and further the exploration of its potential as both a therapeutic target or diagnostic marker.. In this study, we characterized seven SMOC-1 commercial antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified successful antibodies in the tested applications and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Efficient full-length IgG secretion and sorting from single yeast clones in droplet picoreactors.

The search for new antibodies is a major field of pharmaceutical research that remains lengthy and costly due to the need for successive library screenings. Existing in vitro and in vivo antibody discovery processes require that libraries are repeatedly subcloned to switch the antibody format or the secretory host, a resource-intensive process. There is an urgent need for an antibody identification platform capable of screening large antibody libraries in their final soluble format. Previous attempts to develop such a platform have struggled to combine large antibody libraries with screening of high specificity, while retaining sufficient library diversity coverage (ability to detect rare events). Here, we describe a new antibody screening platform based on the encapsulation of antibody secreting yeast cells into picoreactor droplets. We developed and optimized a Yarrowia lipolytica yeast strain capable of growing and secreting full-length human IgGs in picoreactors, and applied a microfluidics-based high-throughput screening approach to sort and recover target-specific antibody-secreting yeasts. Critically, the direct recovery of secretory yeasts allows for downstream screening and antibody characterization, without the need to reformat or subclone the coding sequences. We successfully increased the diversity coverage of sorting the antibody library without compromising sorting specificity by developing a new fluorescence signal processing methodology. By combining this drastically enhanced sorting efficiency with the high-throughput capability of droplet microfluidics, and the rapid growth of Y. lipolytica, our new platform is capable of screening millions of antibodies per day and enriching for target-specific ones in 4 days. This platform will enable the efficient screening of antibody libraries in a variety of contexts, including primary screening of synthetic libraries, affinity maturation, and identification of multi-specific or cross-reactive antibodies.

Antibody Selection via Phage Display in Microtiter Plates.

The most common and robust in vitro technology to generate monoclonal human antibodies is phage display. This technology is a widely used and powerful key technology for recombinant antibody selection. Phage display-derived antibodies are used as research tools, in diagnostic assays, and by 2022, 14 phage display-derived therapeutic antibodies were approved. In this review, we describe a fast high-throughput antibody (scFv) selection procedure in 96-well microtiter plates. The given detailed protocol allows the antibody selection ("panning"), screening, and identification of monoclonal antibodies in less than 2weeks. Furthermore, we describe an on-rate panning approach for the selection of monoclonal antibodies with fast on-rates.

An understanding of the evaluation of histocompatibility testing for a deceased-donor transplant

Dear Editor, Several obstacles have been overcome in the recent decades to make organ transplantation an effective lifesaving treatment for many patients. However, there is a tremendous disparity between the demand for organs and the number of organs necessary around the world, which is regarded as one of the major barriers, and India is not an exception.[1] Since 2013, the rate of deceased-donor organ donation increased from 0.27 per million people (pmp) to 0.65 pmp in 2018. Whereas the rate of deceased-donor renal transplant increased from 0.43 pmp to 0.86 pmp during the same period; this is roughly a 2.4-fold increase in just 5 years.,[2] Still, a lot of support and awareness for deceased-donor organ transplant programs is needed to help patients who do not have living kidney donors. There are numerous obstacles to overcome, with persuading family members being one of the most significant. Furthermore, transportation and allocation of the cadaver are also other issues associated with deceased-donor transplants. Once a cadaver is available, a set of histocompatibility testing of the potential patients is performed to determine the transplant’s suitability. Hence, a transplant immunology laboratory’s ability to accurately report the compatibility tests (to evaluate preformed human leukocyte antigen [HLA] donor-specific antibodies) on time is critical to the success of a deceased-donor transplant program. We want to emphasize the importance of histocompatibility testing evaluation in deceased-donor transplant programs. To support the deceased-donor transplant program, the laboratory must set up an efficient operational system to determine what kind of tests will be needed before, during, and after the transplant. In addition, a functioning 24-h on-call system should be in place. So far, a standalone transplant focused laboratory in North India, accredited for ISO15189: 2012, has aided pretransplant testing workup for 62 cadaveric cases (between February 2015 and December 2022) by optimizing the appropriate and effective test for anti-HLA antibodies between complement-dependent cytotoxicity (CDC) crossmatch, flow cytometric crossmatch (FCXM), and Luminex crossmatch.Both the CDC and the FCXM are time-consuming and labor-intensive, with the potential of false results, especially in the case of deceased donations where there is no time to redo the tests. Even technological advancements have contributed to a better understanding of transplant immunology in the recent years. In the identification of HLA antibodies, the advent of Luminex cross-matching technology provides more sensitivity than CDC and FCXM. Furthermore, unlike Luminex crossmatch, CDC and FCXM are manually created assays that require manual interpretation and whose processing method varies between laboratories. Furthermore, they (CDC and FCXM) both require a high number of viable lymphocytes to complete the experiment, whereas Luminex crossmatch requires a cell lysate to process. It is difficult to isolate a high number of viable lymphocytes from a cadaver; hence, cell count is a major concern in the cadaver transplant workup. The outcome may be harmed as a result of sample quality difficulties. Due to the aforementioned constraints, these approaches are less reliable during cadaver workup, and they also take a longer time than Luminex crossmatch. Even lysate can be saved for posttransplant monitoring.[3,4] Mishra et al. also support the findings of the use of Luminex crossmatch for pretransplant workup of renal transplants.[4] Later on, data published by Mathur et al. on 342 patients supported the usefulness of Luminex lysate crossmatch in deceased donor pretransplant workup.[3] Further, some more published literature[5,6] supports these findings; hence, Luminex crossmatch (lysate) should be used as the primary compatibility investigation for deceased-donor transplants. At present, no specific pretransplant compatibility workup for deceased-donor transplants is available. In routine practice, broadly, the pretransplant compatibility workup falls into two broad categories; cell-based tests (CDC and FCXM) and HLA bead-based (solid phase) tests (such as anti-HLA antibody screening and ant-HLA antibody identification). Laboratories typically utilize testing algorithms for compatibility, which are combinations of two or more tests, since no single test, whether cell-based or HLA bead-based, is 100% accurate and sensitive. To make donor lysate Luminex crossmatch a consensus technique, transplant physicians can be taught about the benefits. Recently, some pretransplant compatibility testing algorithm in the Indian setup is published,[7,8] which necessitates the development of one for better organ transplant outcomes, particularly in cadaver instances where there are numerous hurdles (both clinical and nonclinical). For deceased donor workup, expert analysis in a quicker turnaround time is critical, and hence, the laboratory should be equipped with cutting-edge advanced histocompatibility equipment to aid in the evaluation of histocompatibility testing for deceased-donor transplants. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

The Interference of Monoclonal Anti-Cluster of Differentiation 47 on Transfusion Compatibility Testing

Hu5F9-G4 and AK117, novel cluster of differentiation 47 blockers, are used to treat solid tumors and hematoma. Because cluster of differentiation 47 is expressed on erythrocytes, treatment with monoclonal anti-cluster of differentiation 47 may interfere with the results of transfusion compatibility testing. This study analyzed the interference and treatment of Hu5F9-G4 and AK117 on transfusion compatibility testing. Saline and microcolumn gel methods were used to identify the blood types of the patients. To screen irregular antibodies, salt water, indirect antiglobulin test and polyamine methods were used. Various methods were used to identify antibodies, including saline, indirect antiglobulin test, polyamine and acid diffusion. This paper explores current methods for removing interference caused by monoclonal anti-cluster of differentiation 47. Hu5F9-G4 interferes with reverse typing of ABO blood groups, resulting in agglutination of all reagents used in antibody screening and identification. Patients’ erythrocyte acid dispersive fluid reacted positively with spectrum cells and erythrocytes treated with papain reacted positively with serum. In saline and coagulant amine media, AK117 did not agglutinate with screening cells, but it did with antihuman globulin media. Pre-transfusion testing can be interfered with by anti-cluster of differentiation 47 antibodies. To ensure safe blood transfusions, patients should have their blood type tested and antibodies screened before treatment.

R2 R2 Phenotype Blood Requirements for Liver Transplantation Surgery in a Child with Multiple Rh Antibodies

Red cell alloimmunization often poses challenge for transfusion support during surgery. As transfusion needs are unpredictable in liver transplant recipients, it requires well-equipped immunohematology laboratory for timely antibody identification and good coordination with other blood centers, for the arrangement of compatible units in case of rare phenotypes. We present a case of child with multiple Rh antibodies (Anti-C & anti-e) requiring rare R2R2 phenotype blood units for liver transplantation. With no antigen negative units available in inventory, various blood centers in India were approached individually and through social media. Eleven R2R2 units were transferred by air from three blood centers from North India (New Delhi) to South India (Chennai) with the help of Courier services maintaining cold chain over 24 h with data logger facilities. The patient underwent LT with 2 units (R2R2 phenotype) transfused intraoperatively and 3 units in postoperative period. The patient was discharged on postoperative day 18 with Hb 8.0 gm/dL. The case highlights the need for national/zonal database of rare phenotype blood donors to timely fulfil the blood requirement of patients in need.