Severe allergic and anaphylactic transfusion reactions in consecutive recipients from the same donor

Following multiple allergic/anaphylactic transfusion reactions (AATR) traced to a single donor, plasma-associated Fc-ε-receptor I (FcεRI)-specific immunoglobulin (Ig)G-auto-antibodies were revealed as a rare but potent AATR trigger. AATR are a group of adverse reactions to blood product transfusions where combined donor and recipient factors enhance risk of reactions.¹ Donor-derived causes inducing AATR consist of a heterogenous group of immunoreactive molecules.² Several methods can detect these molecules¹ that clinically, are often focused on specific causative agents such as anti-IgA in IgA-deficient patients or other IgE-dependent pathways.^3-5 Also, AATR often occur in complex clinical settings e.g., trauma centers or perioperative settings involving concurrent drug administration that can cause allergy/anaphylaxis. Therefore, specialized allergy investigation is warranted to exclude drug allergy before consideration of AATR. Amongst newer diagnostic approaches to AATR-detection is the basophil activation test (BAT). Here, donor serum induces degranulation in basophils allowing rapid and sensitive functional assessment regardless of the underlying immunological mechanism.

AATR are rare but underreported, and improved blood establishment detection methods are needed for proper clinical assessment of AATR.⁶ Applying functional assessment tools and characterization of antibodies/antigens involved in AATR could improve recipient outcomes. In addition to identifying multiple life-threatening AATR in recipients of a single donor’s blood, revealed by BAT, we demonstrate how hemovigilance data, donor immunophenotyping, BAT, and combined immunoprecipitation and liquid chromatography mass spectrometry enables advanced AATR assessment. In 2020, two cases of AATR were observed after transfusion of blood products from the same donor. The donor was A RhD^- with 125 prior donations, who, after assessment, was permanently deferred from further blood donation. The blood establishment initiated a 3-year look-back from 2020 to 2017 including assessment of reported AATR and clinical assessment of the donor. The study was done in accordance with General Data Protection Regulation, and the donor gave informed oral and written consent to publication of findings. The study was conducted in alignment with Danish law and adhered to the principles of the Declaration of Helsinki.

Adverse reactions in patients receiving transfusions from the AATR-inducing donor were assessed according to the International Society of Blood Transfusion (ISBT) Hemovigilance Working party including category, severity, and imputability. Patients experiencing AATR received either red blood cells (RBC), fresh frozen plasma (FFP), or platelet components from six donors with platelet additive solution (SSP+, Macopharma, France), but without pathogen reduction/inactivation. Assessment included evaluation of medication administered at the time of potential AATR. In two of the cases, severe anaphylaxis occurred during surgery and full allergy evaluation of all administered drugs was performed without identifying a causative drug. Presence of IgA/anti-IgA antibodies was applied as post-AATR serological assessment. Serum tryptase was available from time of AATR in seven cases, but only confirmed elevated by comparison with baseline samples in two perioperative AATR. For the remainders, imputability was therefore categorized as probable rather than definite.

Hematological profiling of the AATR-inducing donor was analyzed using a Sysmex^® XN on 2020/2022 EDTA plasma and a clinically applied immunodeficiency flow cytometry (FC) panel that was previously reported.⁷ The allergic profile of the donor was assessed with specific IgE assays using ImmunoCAP, BAT and confirmed by basophil histamine-release test.

BAT was performed on plasma and serum samples. Whole blood basophils from healthy individuals were stimulated with healthy donor serum, positive controls (positive: anti-IgE 1 μg/mL; non-releaser: N-formylmethionyl-leucyl-phenylalanine) with parallel analysis of the AATR-inducing donor’s crude serum, IgG-depleted, and serum IgG fraction at varying concentrations. CD63-positive basophils were interpreted as activated. Purified IgG from the AATR-inducing donor was then coupled to beads and incubated with mast cell line (Laboratory of Allergic Diseases 2 [LAD2]) lysate. Precipitated proteins were eluted from antibodies by trypzination. The eluate was analyzed by a trapped ion mobility spectrometry and time-of-flight (timsTOF) Pro mass spectrometer. Raw mass spectrometry data was analyzed with MaxQuant (v1.6.15.0). Statistical analysis of label-free quantification derived protein expression data used the automated analysis pipeline of the Clinical Knowledge Graph.⁸ Relative protein amounts were determined by the MaxLFQ algorithm with a minimum ratio count of two. Mass spectrometry analyses were performed by the Proteomics Research Infrastructure (PRI) at the University of Copenhagen, supported by the Novo Nordisk Foundation (grant number NNF19SA0059305). The mass spectrometry proteomics data was deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange. org) via the PRIDE partner repository⁹ (data set identifier PXD045721).

Recipients and their reaction characteristics are presented in Table 1. The look-back revealed clinical signs of AATR in nine of ten consecutive recipients from 2018 to 2020. Before the first case in 2018, six consecutive recipients experienced no AATR.

Table 1.Recipient allergic/anaphylactic hemovigilance overview.

The AATR-inducing donor reported a distant history of urticaria on skin exposure to grass and had received subcutaneous grass allergy immunotherapy. There was no history of allergic reactions, unusual infections, surgery or transfusion in the period prior to 2018. Overall, the clinical work-up was unremarkable: normal overall T-, B-, and NK-cell concentration including subpopulations and major myeloid cell subpopulations; normal Ig concentrations and complement function. Screening for anti-HLA/-HPA/-HNA-antibodies was negative, but neutrophil agglutination after exposure to donor serum was observed with 1 of 4 neutrophil-assay donors. The only finding in donor’s hematological profile was extreme basopenia between 2010 and 2022, which was confirmed using FC basophil markers CRTH2/CD123 (Figure 1A). Underlying hematological disease was excluded via adiagnostic leukemia FC panel. The AATR-inducing donor’s serum was highly positive using basophil histamine-release test. Skin prick test (SPT) with histamine and increasing morphine doses¹⁰ was positive. Combined with normal baseline serum tryptase, this confirmed the presence of functional skin mast cells.

Figure 1.Serum from an AATR-inducing donor activates basophils from healthy controls. (A) Representative dot plots showing CD63⁺ basophils in healthy controls and the allergic/anaphylactic transfusion reactions (AATR)-inducing donor, unstimulated and after anti-immunoglobulin (Ig)E stimulation. (B) Degranulation of basophils from healthy controls following stimulation with serum or plasma from the AATR-inducing donor. (C, D) Degranulation of basophils from healthy control (Ctrl) stimulated with (C) serum from a healthy donor versus AATR-inducing donor (2020, 2022) (D) crude, IgG, and IgG-depleted serum from the AATR-inducing donor. (E) Degranulation of healthy control basophils following incubation with archival plasma (2013–2022) from the AATR-inducing donor, annotated with clinical reactions. BD: basophil degranulation.

Figure 2.Functional and proteomic analyses indicate FcεRI as the target of AATR-donor autoantibodies. (A) Degranulation of basophils from healthy controls following stimulation with healthy and allergic/anaphylactic transfusion reactions (AATR)-sera depleted of autoantibodies by incubation/ elution of sera with Fc-ε-receptor I (FcεRI)-expressing cells (LAD2, KU812) and immuno-globulin (Ig)E-coated beads. (B) Volcano plot of combined immunoprecipitation and mass spectrometry results showing relative abundance of proteins targeted by AATR-donor antibodies. Donor IgG was incubated with lysate of LAD2 cells followed by immunoprecipitation. Comparison of donor IgG to control IgG from healthy individual is displayed. X-axis shows log2-fold change (Log2FC) with red-labeled proteins denoting increased Log-2FC of proteins targeted by donor IgG, while blue-labeled show decreased Log2FC. Y-axis shows the negative log10 P value. Proteins with no Log2FC differences between index donor sample and healthy controls or proteins that did not reach statistical significance are represented by grey dots.

EDTA and heparinized plasma, as well as serum from the donor, induced comparable, high-level degranulation of basophils from healthy individuals (Figure 1B). Reactivity of donor’s serum from 2020 was 5% higher compared to 2022, showing 87% versus 82% of CD63-positive basophils. Surprisingly, dilution of crude serum showed that even a 0.06% concentration induced degranulation of basophils (25%) (Figure 1C). To determine the serological trigger of the response, donor’s serum was depleted of IgG and tested on basophils from healthy individuals. The isolated IgG fractions induced responses comparable to crude serum, while the IgG-depleted fractions had no effect, indicating IgG antibodies as the causative agent of AATR (Figure 1D). Randomized analysis of archival plasma samples from the donor including samples with evident clinical AATR (N=3) and from before onset of reactivity in 2018 (N=18) revealed positive degranulation in only the post-2018 samples (Figure 1E).

Since the basophil response to the AATR-inducing serum was comparable to anti-IgE activation, we assumed that the FCεRIα pathway was targeted by the IgG antibodies in the donor’s serum. After preincubation of serum with either FcεRIα-expressing LAD2 cells, KU812 cells, or IgE-positive microbeads, only sera from the LAD2 cells showed reduced capacity to activate basophils, indicating that FcεRIα, rather than IgE, was the IgG-antibody target (Figure 2A). Following immunoprecipitation of antibody/antigen complexes after incubation of the donor’s serum with LAD2 cells, mass spectrometry analysis could identify antibodies directed against both α- and |3-subunits of FceRI (FCER1A, MS4A2) with manifold increased detection compared to a healthy control (Figure 2B).

In conclusion, multiple consecutive severe AATR had occurred in recipients who received donations from a single blood donor, that had remained undetected by standard clinical measures for AATR detection for several years. The chance finding of two transfusion reactions to blood components from the same donor led to a large scale look-back revealing seven additional cases. Despite research suggesting BAT as a screening tool, no international recommendations exist regarding prevention of AATR or donor screening.

Milder AATR were observed in patients receiving RBC components, which contain less plasma than platelets or FFP also implying the causative mechanism as plasma associated. Here, we suspect a FceRI-specific IgG autoantibody in the AATR-inducing donor’s plasma/serum as the causative mechanism. The presence of anti-FceRI autoantibodies is well known in patients with chronic spontaneous urticaria (CSU).¹¹ One possible etiology of the AATR-inducing mechanism could be subclinical CSU in the AATR-inducing donor, resulting in the extreme potency of the donor’s serum/plasma in inducing basophilic degranulation. Another mechanism could be an undetected/occult infection leading to immune activation triggering the change in reactivity, possible mediated by somatic hypermutation and affinity maturation.¹² Population studies have found that IgG directed against FcεRI are prevalent in both patients with CSU and healthy controls,¹³ which could complicate large-scale blood donor screening for AATR-inducing FcεRI antibodies if parallel functional assessment is omitted.

Clinical AATR-signs are difficult to distinguish from allergic reactions to concurrently administered medication, which are more common and should be investigated first. Identifying AATR-inducing donors through clinical reporting and backwards traceability of AATR seems insufficient. The addition of high-throughput functional assays could help to screen donor populations for prevalence of AATR induction. Our results highlight the possibility of identifying causal AATR-mechanisms by going beyond BAT. While we acknowledge this level of workup cannot be performed on all donors, this approach may enable in-depth immunological assessments to identify the few but relevant donors responsible for AATR, with expected benefits for recipient safety. This study illustrates a blood establishment approach to performing clinical assessments of donors involved in AATR. This is an area that would benefit from international consensus guidelines, which we urge the international hemovigilance community to consider.

Footnotes

• Received August 6, 2025
• Accepted November 19, 2025

Correspondence

*PTB and CM contributed equally as first authors

P.T. BROOKS - patrick.terrence.brooks@regionh.dk

Disclosures

No conflicts of interest to disclose.

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