Detection Of Alloantibodies Research Articles

In vitro Generated Megakaryocytes for the Detection of Human Platelet Antigen-Specific Alloantibodies

Plain Language SummaryThe identification of antibodies to human platelet antigens (HPA) is crucial. The standard test (MAIPA assay) is difficult because it requires fresh platelets from specific donors. This study investigated a new method, monoclonal antibody immobilization of megakaryocyte antigens (MAIMA), using in vitro generated megakaryocytes from CD34+ cells. The assay was tested against WHO reference reagents and patient samples with known anti-HPA antibodies. Results showed that MAIMA successfully detected anti-HPA-1a antibodies and correlated well with MAIPA. However, MAIMA had lower sensitivity for anti-HPA-3a and anti-HPA-5b antibodies. Notably, anti-HPA-5b antibodies were not detectable by MAIMA. In conclusion, in vitro generated megakaryocytes can effectively detect anti-HPA-1a antibodies. However, MAIMA may be less suitable for antibodies against other HPAs, such as HPA-5b. This study highlights the potential of MAIMA as an alternative assay and provides insight into its strengths and limitations in detecting specific anti-HPA antibodies.

A national survey of current immunohematologic testing practices for the diagnosis of autoimmune hemolytic anemia in India.

Autoimmune hemolytic anemia (AIHA) is a common term for several disorders that differ from one another in terms of etiology, pathogenesis, clinical features, and treatment. Management of patients with AIHA has become increasingly evidence-based in recent years. While this development has resulted in therapeutic improvements, it also carries increased requirements for optimal diagnosis using more advanced laboratory tests. Unfortunately, limited data are available from developing countries regarding the testing and transfusion management of patients with AIHA. The main objective of this survey was to explore the current immunohematologic testing practices for the diagnosis of AIHA in India. This online survey consisted of 30 questions, covering the place of work, the number of AIHA cases encountered in the 3 preceding years, testing method(s), transfusion management, and so forth. Individuals representing 89 laboratories completed the survey; only 78 of which responded that AIHA testing was performed in their facility's laboratory. The majority of respondents agreed that the most commonly affected age-group comprised individuals of older than 20 years, with a female preponderance. Regarding transfusion management, respondents indicated that transfusion with "best-match" red blood cell units remains the most common practice. Column-agglutination technology is used by 92 percent of respondents as the primary testing method. Although a monospecific direct antiglobulin test is available at 73 percent of the sites, most of them have limited access to other resources that could diagnose cold or mixed AIHA. Merely 49 percent of responding laboratories have the resources to perform adsorption studies for the detection of alloantibodies. Furthermore, three-cell antibody screening reagents are unavailable at 32 percent of laboratories. In 72 percent of centers, clinical hematologists would prefer to consult a transfusion medicine specialist before administering treatment to AIHA patients. There is unanimous agreement regarding the need for a national registry. The survey data indicate wide variability in testing practices for patients with AIHA in India. Future studies are needed to focus on the feasibility and cost-effectiveness of different testing strategies for developing countries.

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

Current challenges of blood transfusions in patients with thalassemia in India and future perspectives

The introduction of regular red blood cell transfusions transformed thalassemia major from a fatal childhood disease into a chronic disorder. Thalassemia is highly prevalent in South Asia, including the Indian subcontinent, and blood transfusion remains the cornerstone of management for these patients. But safe blood transfusions still remain a major problem in India. Difficulties in maintaining adequate blood inventory, a lack of a national blood act, and fragmented blood transfusion services are some of the major contributing factors for the delay in blood supply. In most of the blood centers, alloantibody detection facilities and extended red cell antigen typing are unavailable. Awareness is the key to reducing alloimmunization, which limits the effectiveness of transfusions and the potential availability of blood. Patients with thalassemia are also at high risk of transfusion-transmitted infections unless appropriate blood screening is in place. Hence, many patients remain under-transfused, resulting in decreased health and quality-of-life outcomes. Facilities such as leucoreduction and immunohematological monitoring following a blood transfusion are often lacking in India, especially at the sub-district level. Continuous efforts to raise community awareness, regular training of health-care workers, and proper utilization of available resources are essential to ensuring safe blood transfusions for patients with thalassemia. Access to the new treatments at an affordable cost may reduce the blood transfusion burden for thalassemia patients in India.

The importance of removing interference caused by Darzalex® (daratumumab) in pre-transfusion testing

Introduction: Multiple myeloma (MM) is a hematologic malignacy characterized by the uncontrolled proliferation of abnormal plasma cells in the bone marrow. Darzalex® (daratumumab) has emerged as a highly effective therapeutic agent for MM, offering an additional treatment option for patients who have developed resistance to other therapies. However, the use of Darzalex® poses challenges in pre-transfusion testing due to its strong binding affinity of the monoclonal anti-CD38 antibody to CD38 protein, resulting in panreactivity with test erythrocytes and complicating the detection of erythrocyte alloantibodies. The objective of this case report is to outline the procedures, significance, and methodologies for mitigating this interference using the DaraEx® reagent. Case Report: We present the cases of three patients diagnosed with MM and treated with Darzalex® at our institution in 2023. Following the administration of Darzalex®, all three patients exhibited positive results on indirect antiglobulin tests (IAT). Cross-matching tests conducted with red blood cell concentrates also yielded positive results, with strengths ranging from 2+ to 3+. To eliminate interference, we used a specific DaraEx® reagent that utilizes Fab fragments of anti-CD38 to mitigate interference by masking CD38 on the cell surface. Subsequent application of this reagent resulted in negative IAT results for all three patients. However, one patient still displayed a weakly positive cross-matching test (<1+), suggesting that complete elimination of interference may not always be achievable with the DaraEx® method. Conclusion: Despite the availability of reliable methods such as dithiothreitol and the DaraEx® reagent for mitigating interference, complete success in eliminating interference remains elusive. This underscores the importance of implementing proactive measures to prevent interference. Patients undergoing Darzalex® therapy should undergo comprehensive immunohematological testing before medication administration. Given the increased need for blood transfusions in MM patients, ensuring the compatibility of blood products is essential for safe transfusion practices.

Significance of red cell alloantibodies other than anti-D during pregnancy and their effect on the newborn: A case series

Contrary to well-established guidelines in developed countries, awareness regarding red cell alloantibodies in antenatal period are lacking in India. Investigating for indirect antiglobulin test (IAT) is mostly limited to the Rh D negative antenatal cases. This case series revisits this vital aspect of maternal and fetal safety. Instances of alloantibody other than anti-D are reported.Study was done in Transfusion Medicine department of a tertiary care hospital in North India during 2019-2020. IAT was performed not during the 1st or 2nd trimesters of pregnancy but as a routine compatibility test during delivery. Patients with positive IAT were further evaluated for the detection of alloantibody by using identification panel red cells. Result: Eight antenatal cases with irregular antibodies other than anti-D during 2019-2020 are described. Antibodies detected per patient were single (three cases of anti-E, one of anti-Fya, one of anti-M) or multiple (two cases of anti-E plus anti-c, one of anti-E plus anti-K). Direct antiglobulin test of four babies born to these mothers was found to be positive, one of whom was still born and rest recovered with medical management. Two other babies had DAT negative and two mothers presented late after still birth. Alloantibody titer indicated in patient with anti-E during mid-pregnancy had titer was undetectable by standard tube technique.Non anti-D alloantibodies can potentially affect fetus, asserting equal attention as anti-D. IAT should not be missed in pregnancy as it is common to investigations for compatibility as well as for fetal wellbeing assessment.

On a Long and Winding Road: Alloantibodies in Organ Transplantation.

Today we know that both the humoral and the cellular arm of the immune system are engaged in severe immunological challenges. A close interaction between B and T cells can be observed in most "natural" challenges, including infections, malignancies, and autoimmune diseases. The importance and power of humoral immunity are impressively demonstrated by the current coronavirus disease 2019 pandemic. Organ transplant rejection is a normal immune response to a completely "artificial" challenge. It took a long time before the multifaceted action of different immunological forces was recognized and a unified, generally accepted opinion could be formed. Here, we address prominent paradigms and paradigm shifts in the field of transplantation immunology. We identify several instances in which the transplant community missed a timely paradigm shift because essential, available knowledge was ignored. Moreover, we discuss key findings that critically contributed to our understanding of transplant immunology but sometimes developed with delay and in a roundabout way, as was the case with antibody-mediated rejection-a main focus of this article. These include the discovery of the molecular principles of histocompatibility, the recognition of the microcirculation as a key interface of immune damage, the refinement of alloantibody detection, the description of C4d as a footmark of endothelium-bound antibody, and last but not least, the developments in biopsy-based diagnostics beyond conventional morphology, which only now give us a glimpse of the enormous complexity and pathogenetic diversity of rejection.

Comparing red cell alloantibody detection and titration by gel microcolumn agglutination method with conventional tube in Rh-negative antenatal cases

BackgroundAlloimmunization to red cell antigens during antenatal period is a serious complication. Antibody titration of maternal blood sample corroborates with severity of disease and helps in planning further management. Conventional Tube Testing (CTT) method is widely accepted for antibody titration in antenatal cases but it can be replaced by Gel Microcolumn Agglutination (GMA) as it offers many advantages. Critical titer levels have been recommended by CTT method, but corresponding levels by GMA are yet to be established due to lack of adequate data. MethodsTotal of 1020 Rh negative antenatal females were evaluated over a period of four years and those found to be positive on antibody screening were further investigated. Antibody titration by CTT and GMA was carried out and the titers were correlated with the outcome of pregnancy. ResultsOut of the 1020 cases screened for antibodies, 112 (10.98%) were detected to be positive by GMA while 40 (3.92%) cases were detected by CTT. Titration was performed by both methods and results were statistically correlated. A moderate correlation was observed where one or more than one antibodies were involved (Pearson’s Coefficient 0.756), while a strong correlation (0.893) was observed between the two methods when single antibody (Anti-D) was involved. Titer values obtained by GMA were also higher than CTT. ConclusionGel microcolumn agglutination (GMA) was found to be more sensitive and precise method for titration. A titer of 512 by GMA (IgG only) mostly corresponded to the values of 16 by CTT and had good correlation with clinical outcome.

Laser incubation for the rapid detection of red cell alloantibodies in human blood samples.

Pre-transfusion antibody screening requires the detection and identification of immunoglobulin G (IgG) antibodies against red blood cells (RBCs). Using the indirect antiglobulin test (IAT), plasma-RBC solutions are incubated at 37°C in gel cards, typically by heating block technology. Here, we apply the newly developed laser incubation method to detect RBC alloantibodies in the plasma from human donors. Donated human plasma samples (N=128) containing clinically significant IgG antibodies directed against Rh (D, C, c, Cw and E), Kell (K and Kpa ), Duffy (Fya and Fyb ), Kidd (Jka ) and MNS (S) blood group system antigens were tested by the indirect antiglobulin test (IAT). Samples were heated to 37°C by infrared laser (980 nm) for incubations of up to 5min. Samples were also incubated in a heating block for comparison. When heating by laser, the presence of an alloantibody is detected after only a 1-min incubation for 96% of samples. No samples required longer than 3min of laser incubation in order to detect the antibody. For all samples, incubation by laser gave the same or stronger result within 5min. No samples required longer than 5min to achieve an equivalent result to that of the 5-min heating block incubation. The laser was not found to damage cells or antibodies. Laser incubation provides comparable results in shorter time frames than the heating block. Laser incubation can rapidly detect even very weak antibodies.

Efficient neutralization of daratumumab in pretransfusion samples using a novel recombinant monoclonal anti-idiotype antibody.

Anti-CD38 antibodies such as daratumumab (DARA) are critical therapies for multiple myeloma and other diseases. Unfortunately, anti-CD38 antibodies cause panreactivity in indirect antiglobulin tests (IATs), complicating blood compatibility testing. The anti-CD38 interference is most often mitigated by treating reagent red blood cells (RBCs) with dithiothreitol (DTT). However, when using the DTT method, not all RBC antibody specificities can be detected (e.g., anti-K), and the DTT method is impractical for some transfusion services. We evaluated the ability of a new anti-idiotype antibody to neutralize DARA in vitro and eliminate the anti-CD38 interference. A recombinant monoclonal rabbit anti-DARA idiotype antibody ("anti-DARA") was generated. The ratio of anti-DARA required to neutralize DARA in spiked samples was evaluated in IATs performed in gel. IATs performed in tube were used to demonstrate that anti-DARA allows alloantibody detection in the presence of DARA. Plasma samples from 29 patients receiving DARA were treated with a fixed quantity of anti-DARA (120 μg) before performing antibody detection tests (screens) in tube. Anti-DARA used at or above a 1:1 ratio with DARA eliminated the DARA interference with IATs. Anti-DARA allowed detection of both alloanti-E and alloanti-K in the presence of DARA. In 27/29 (93.1%) clinical samples, 120 μg anti-DARA was sufficient to neutralize the DARA in 100 μl patient plasma. An anti-DARA:DARA ratio as low as 1:1 is sufficient to neutralize DARA in solution. A fixed amount of anti-DARA eliminates the anti-CD38 interference in most patient samples.

Detection of alloimmunization in Glanzmann Thrombasthenia and Bernard-Soulier Syndrome: Data from a Brazilian Center

IntroductionThe Glanzmann Thrombasthenia (GT) and Bernard-Soulier Syndrome (BSS) are rare hereditary disorders of platelet function. Their treatment often requires platelet transfusion, which can lead to the development of alloantibodies. ObjectiveIn this study, we aim to develop a strategy for alloantibody detection and to describe the frequency of alloimmunization in a patient population from a single center in southeastern Brazil. MethodsSamples from patients with GT or BSS were tested using the Platelet Immunofluorescence Test (PIFT). If a positive result was obtained, a confirmatory step using the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) and Luminex bead-based platelet assay (PAKLx) was executed. Main results: Among 11 patients with GT, we detected the presence of alloantibodies in 5 using PIFT, with confirmation through MAIPA and PAKLx in 2 (1 anti-HLA and 1 anti-HPA), resulting in a frequency of 18.1%. Among 4 patients with BSS, PIFT was positive in 3, with confirmation by MAIPA and PAKLx in 1 (anti-HLA), showing a frequency of 25%. The two patients with anti-HLA antibodies exhibited a panel reactive antibody (PRA-HLA) testing greater than 97%. ConclusionOur study highlights the importance of identifying platelet alloimmunization in this patient population. The proposed algorithm for platelet alloantibodies detection allows resource optimization.

Red cell alloimmunization in transfusion-dependent β-thalassemia patients attending Alexandria University Children Hospital

Background In Egypt, it has been established that the most frequently inherited disorder of hemoglobin is β-thalassemia. Management of the severe forms of this disorder consists chiefly of red cell transfusion. The concerned patients receive these transfusions on a regular basis. Therefore, the formation of antired cell alloantibodies and autoantibodies was inevitable. This dilemma is addressed in this study that aims to determine the prevalence of red cell alloimmunization among transfusion-dependent ß-thalassemia children that receive frequent care in the Hematology Clinic of Alexandria University Children’s Hospital. Patients and methods This study screened 100 transfusion-dependent patients in total, all of them were 3–16 years old. Transfusion and clinical records of the latter were reviewed to assess the diagnosis of β-thalassemia, the age of the first presentation, the age at which transfusion therapy was started, transfusion interval, and the number of red cell units given. Alloantibody detection was then performed. Results In the present study, 11 (11%) out of the total 100 studied patients were alloimmunized. The total number of alloantibodies identified in these patients was 25. Two patients (18%) had only one alloantibody, six patients (54.5%) had two alloantibodies, one patient (9%) had three alloantibodies, and two patients (18%) had more than three alloantibodies. The most prevalent antibodies belonged to Kell and Rh blood-group systems (seven out of 25 each). Conclusions When it comes to thalassemia patients dependent on blood transfusion, red blood cell alloimmunization is a difficult complication. Kell and RH blood-group-system antibodies are the predominant antibodies. Formation of alloantibodies depends mainly on the nature of red cell units received and donor and recipient factors. These can explain the difference in alloimmunization rates in different reports. Avoidance of alloimmunization would decrease the cost of treatment on the long run and improve the patient quality of life.

Effective Neutralization of Daratumumab Effects on Pre-Transfusion Testing: a Method Modification.

The anti-CD38 antibody daratumumab is a common multiple myeloma treatment. As the erythrocyte's membrane expresses CD38, Daratumumab-treated samples show agglutination in serological pre-transfusion tests, hindering detection of erythrocyte alloantibodies. Dithiothreitol interferes with erythrocyte antigens, affecting investigation of unexpected antibodies. DARAEx®, an anti-CD38 neutralizing agent, overcomes daratumumab-induced effects, without dithiothreitol's interferences. DARAEx® is applied only in Biorad columns. This study aimed to provide a DARAEx® protocol for application with the Grifols platform. We introduced a modified DARAEx® protocol (AssutaBB protocol) and performed antibody screenings on samples from nineteen daratumumab-treated patients. The AssutaBB protocol provided antibody screen results for all patients, exactly as established in the default manufacturing protocol. Eleven patients presented natural negative antibody screens; eight presented positive K/E antibodies. AssutaBB allows the use of the more widespread Grifols platform in daratumumab-treated patients.

Genetic Removal of Terminal Sialic Acid Residues from the O-Linked Glycans Adjacent to the HPA-9b Polymorphism of Platelet Membrane Glycoprotein IIb Improves the Binding and Detection of HPA-9b Patient Alloantibodies

Sialic acids occupy the terminal position of glycan chains, and have the potential to influence the antigenicity of glycoproteins. Antibody binding sites on a glycoprotein can be solely protein in nature, or can include or be affected by nearby glycan chains, which may either mask the epitope, or conversely comprise part of the antibody binding site. The polymorphisms responsible for formation of the Human Platelet Alloantigens (HPA)-3 (Ile843Ser) and HPA-9 (Val837Met) are next to each other near the C-terminus of the extracellular domain of platelet membrane glycoprotein (GP)IIb, and are adjacent to sialyl-Core 1 O-glycans that emanate from serines 845 and 847. Previous studies have shown that these O-linked glycans are required to support the binding of some of HPA-3a alloantibodies. Loss of these glycans, especially terminal sialic acid residues, during platelet storage or preparation, can present major difficulties in detecting clinically important anti-HPA-3a alloantibodies in suspected cases of fetal/neonatal alloimmune thrombocytopenia (FNAIT). Similarly, detection and identification of anti-HPA-9b alloantibodies from FNAIT patient sera can also be extremely challenging, resulting in the inability to resolve clinical cases of this bleeding disorder. Whether the nearby O-glycans on serines 845 and 847 of GPIIb affect the antigenicity of HPA-9b, and/or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of FNAIT is unknown.We previously reported the generation of bioengineered, HLA class I-negative, HPA-9a or -9b allele-specific megakaryocytes (MKs) from human induced pluripotent stem cells (iPSCs) that are suitable for whole-cell flow cytometric detection of anti-HPA-9b alloantibodies (Blood 2019;134(22):e1-e8). Unexpectedly, treatment of these allele-specific MKs with neuraminidase actually enhanced the binding of anti-HPA-9b alloantibodies, suggesting that terminal sialic acids on GPIIb partially mask the HPA-9b epitope. To test the hypothesis that removal of terminal sialic acids on nearby O-glycans, or removal of the entire O-glycan chains emanating from Ser845/847 of GPIIb, might enhance the detection of anti-HPA-9b patient alloantibodies, we created a series of deletion mutants in two major sialidases, ST3GAL1 and ST3GAL2, known to be responsible for transferring terminal sialic acid residues to Core 1 O-glycans, in our HPA-9a and -9b allele-specific iPSCs. Immunoprecipitation/western blot analysis confirmed the complete removal of terminal sialic acids on the O-glycan chains of GPIIb in ST3GAL1/2 knockout (KO) iPSC-derived MKs, as reported by the binding of the lectin PNA to the exposed Core 1 structure on GPIIb. These sialylation-deficient ST3GAL1/2 KO HPA-9b MKs exhibited dramatically increased anti-HPA-9b alloantibody binding, further confirming the notion that HPA-9b epitopes are partially masked by terminal sialic acids on nearby GPIIb O-glycan chains. Finally, allele-specific iPSCs lacking the complete O-glycan chains attached to serines 845 and 847 of GPIIb were generated by mutating those residues to alanines using a similar CRISPR/Cas9 gene editing approach. Interestingly, O-glycan chain-deficient Ala845/847 mutant MKs carrying the HPA-9b polymorphism exhibited slightly to moderately reduced binding of anti-HPA-9b alloantibodies, indicating that the presence of the Core 1 O-glycan chains attached to GPIIb serine residues 845 and 847 contribute to the presentation of the HPA-9b epitope - perhaps by stabilizing the conformation of the glycoprotein in this region. Taken together, these data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of iPSC-derived HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues, but retain the glycan chains to which they are attached. DisclosuresCurtis: Rallybio: Consultancy. Newman: Rallybio: Consultancy, Research Funding.

Corneal Allografts: Factors for and against Acceptance.

Cornea is one of the most commonly transplanted tissues worldwide. However, it is usually omitted in the field of transplantology. Transplantation of the cornea is performed to treat many ocular diseases. It restores eyesight significantly improving the quality of life. Advancements in banking of explanted corneas and progressive surgical techniques increased availability and outcomes of transplantation. Despite the vast growth in the field of transplantation laboratory testing, standards for corneal transplantation still do not include HLA typing or alloantibody detection. This standard practice is based on immune privilege dogma that accounts for high success rates of corneal transplantation. However, the increasing need for retransplantation in high-risk patients with markedly higher risk of rejection causes ophthalmology transplantation centers to reevaluate their standard algorithms. In this review we discuss immune privilege mechanisms influencing the allograft acceptance and factors disrupting the natural immunosuppressive environment of the eye. Current developments in testing and immunosuppressive treatments (including cell therapies), when applied in corneal transplantation, may give very good results, decrease the possibility of rejection, and reduce the need for retransplantation, which is fairly frequent nowadays.

To Reflex or Not to Reflex: A Time and Cost-Effectiveness Analysis of Autocontrol with Reflex DAT versus Direct DAT.

Performing autocontrol with a reflex direct antiglobulin test (DAT) or directly performing IgG DAT only for alloantibody detection has been a matter of institutional preference. The aim of this study is to evaluate antibody identification (ABID), local cost, and staff time savings of both processes. We retrospectively reviewed all positive indirect antiglobulin tests with corresponding ABID, DAT, autocontrol, and patients with newly identified antibodies in 2014 and 2016. The number of tests performed, ABID, and the cost differences between methods were compared. Cost analysis was estimated from direct material costs, labor costs, and time spent per ABID workup. Annual costs and time saved by performing direct IgG DAT only were $8460 and 180 hours, respectively. The percentage of new ABID in 2014 and 2016 was identical (3.3%). Removing autocontrol with reflex DATs at our center reduced costs and staff time while maintaining a comparable rate of positivity of ABID.

Immunohematology and Transfusion Support of a Patient with Acute Myeloid Leukemia and an Anti-Vel

BackgroundTransfusion support of cancer patients presenting with red blood cell (RBC) antibodies against high-incidence antigens is a challenge for hospital-based Transfusion Services. For patients with antibodies against the high-frequency Vel antigen, only 0.0004% of blood donors will be Vel antigen-negative. Anti-Vel is notorious for looking like a benign, cold-reactive IgM antibody or a “high-titer, low-avidity” antibody; however, Vel-antibodies can fix complement at body temperatures and can cause severe hemolytic transfusion reactions. The immunohematology workup and RBC transfusion support are reported for a patient who developed an anti-Vel and other RBC antibodies during chronic transfusion therapy for myelodysplastic syndrome, with progression to acute myeloid leukemia. She was also evaluated for hematopoietic stem cell transplantation (HSCT).MethodsImmunohematologic workups were performed at a regional immunohematology reference laboratory. Since the patient had a positive autocontrol and a positive direct antiglobulin test (DAT), allogeneic adsorptions were performed at 37°C using R1R1, R2R2 and rr RBC reagents. Adsorbed plasma and eluates from adsorbed RBCs were then tested for alloantibody detection using manual tube method at both LISS and PEG reactivity. In preparation for possible HSCT, RBC alloantibody titers and monocyte monolayer assay (MMA) were performed. All samples were collected with pediatric phlebotomy tubes whenever possible.ResultsThe patient was a 66 year old woman with a myelodysplastic syndrome that transformed to acute myeloid leukemia (AML) with complex karyotype a year later. The RBC reference workup identified alloantibodies reacting to the high-incidence Vel antigen as well as low-incidence Yt(b) and Cw antigens. The patient's plasma was non-reactive with three of four Vel-negative (-) cells, whereas the reactive cell was Cw positive (+) and Yt(b+). Two allo-adsorptions were performed to remove anti-Vel. Upon repeat testing, anti-Cw was identified by LISS indirect antiglobulin test. Eluate from adsorbing RBCs was reactive with Vel- and Yt(b+) cells, suggesting presence of anti-Ytb. In preparation for HSCT, anti-Vel and anti-Cw were titered at 0 and 64, respectively. MMA for anti-Vel ranged at 0.2-2% (normal <3%). Nevertheless, the hospital-based Transfusion Service provided rare ABO/Rh Vel- CW- Yt(b-) crossmatch-compatible RBC to minimize the potential for stimulating anti-Vel, which might compromise a Vel+ HSCT. The patient required about 1 RBC unit per week to maintain hemoglobin above 4.5 g/dL. The Transfusion Service coordinated a search for antigen-negative RBCs with a national rare donor program, allowing at least 4 rare RBC units to be available at all times. Over an 18-month time period, the patient received 62 RBC Vel- units: 45 out of 62 (73%) were Cw- and 17 were Cw-unscreened but C-. Later in the patient's course, her transfusion rate increased to 2 RBC units per week. Because insufficient Vel- units were available within the USA, an international search by a rare donor program identified Vel- donors in Europe. FDA approval was obtained to import the rare units. She received 2 units from Spain and 2 units from Switzerland. Her leukemia progressed, and transfusion support was discontinued.ConclusionCoordination between hospital-based Transfusion Service, hematologists, and rare donor programs is required for rare RBC transfusion support in patients with antibodies against high-incidence antigens. A transfusion threshold of 4.5 g/dL may be tolerated when availability of rare RBC units is limited. DisclosuresNo relevant conflicts of interest to declare.

A first WHO reference reagent for the detection of anti-human platelet antigen-15b.

Background and ObjectivesAlloantibodies to human platelet antigen‐15b (anti‐HPA‐15b) have been detected in mothers with foetal–neonatal alloimmune thrombocytopenia and in multiply transfused patients. Assays used to detect this antibody, which aids in disease diagnosis, can be unreliable and vary in sensitivity. The objective was to generate a stable, lyophilized anti‐HPA‐15b preparation and evaluate its suitability as a World Health Organization (WHO) reference reagent for use in the quality control of platelet alloantibody detection assays. Results from an international collaborative study to evaluate the preparation were used to assign a minimum potency at which laboratories can be expected to detect the antibody.Materials and MethodsRecalcified plasma containing anti‐HPA‐15b was aliquotted, lyophilized and coded 18/220. Twenty‐five laboratories in 16 countries tested doubling dilutions of the reconstituted material in glycoprotein‐specific assays such as the monoclonal antibody–specific immobilization of platelet antigen assay and reported the last positive (or endpoint) dilution.ResultsTwenty‐four laboratories (96%) detected antibodies with HPA‐15b specificity in preparation 18/220. Reported endpoint dilutions were normally distributed with a modal dilution of 1 in 16 and ranged from 1 in 2 to 1 in 128. Only two laboratories (8%) failed to detect anti‐HPA‐15b at 1 in 8 dilution.ConclusionsWhen diluted 1 in 8, most laboratories detected anti‐HPA‐15b in preparation 18/220 using HPA‐15bb platelets but not with HPA‐15aa platelets. The participants agreed this to be an appropriate dilution for assignment as the minimum potency. In October 2020, the WHO Expert Committee on Biological Standardization approved 18/220 as an International Reference Reagent.

Pre-transplant donor-reactive IL-21 producing T cells as a tool to identify an increased risk for acute rejection

Pre-transplant screening focuses on the detection of anti-HLA alloantibodies. Previous studies have shown that IFN-γ and IL-21 producing T cells are associated with the development of acute rejection (AR). The aim of this study, was to assess whether pre-transplant donor-reactive T cells and/or B cells are associated with increased rejection risk. Samples from 114 kidney transplant recipients (transplanted between 2010 and 2013) were obtained pre-transplantation. The number of donor-reactive IFN-γ and IL-21 producing cells was analyzed by ELISPOT assay. The presence of donor specific antibodies (DSA) was also determined before transplantation. Numbers of donor-reactive IFN-γ producing cells were similar in patients with or without AR whereas those of IL-21 producing cells were higher in patients with AR (p = 0.03). Significantly more patients with AR [6/30(20%)] had detectable DSA compared to patients without AR [5/84(5.9%), p = 0.03]. Multivariate logistic regression showed that donor age (OR 1.06), pre-transplant DSA (OR 5.61) and positive IL-21 ELISPOT assay (OR 2.77) were independent predictors of an increased risk for the development of AR. Aside from an advanced donor-age and pre-transplant DSA, also pre-transplant donor-reactive IL-21 producing cells are associated with the development of AR after transplantation.