Adverse effect of donor-specific anti-human leukocyte antigen (HLA) antibodies directed at HLA-DP/-DQ on engraftment in cord blood transplantation.

BACKGROUND AIMS: While donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) in the recipient before transplantation are associated with graft failure in cord-blood transplantation (CBT), effects of DSAs other than against HLA-A, -B or -DRB1 on transplantation outcomes remained poorly understood. METHODS: We retrospectively analyzed 567 single-unit CBT recipients to evaluate impact of DSAs against HLA-DP and -DQ on CBT outcomes. RESULTS: Among 143 recipients (25.2%) who had anti-HLA antibodies, nine harbored DSAs against HLA-DP or -DQ. DSAs against HLA-DP or -DQ were associated with a significantly lower neutrophil engraftment rate (55.6% versus 91.8%, P = 0.032) and with a marginally lower platelet engraftment rate (46.7% versus 75.3%, P = 0.128) at day 100 after transplantation, compared with patients without anti-HLA antibodies. Time to neutrophil and platelet engraftment in patients with DSAs for HLA-DP or -DQ was significantly longer than that in patients without anti-HLA antibodies (median, 25 versus 21 days, P = 0.002 in neutrophil; median 61 versus 46 days, P = 0.014 in platelet). Cumulative incidence of bacterial infection at day 100 was significantly greater (88.9% versus 57.1%, P = 0.024), and re-transplant-free survival was marginally lower (55.6% versus 76.8%, P = 0.132) in patients with DSAs against HLA-DP or -DQ, compared with those without anti-HLA antibodies. These findings suggest that DSAs against HLA-DP or -DQ lead to unfavorable engraftment, which may increase risk of bacterial infection, and reduce survival soon after CBT. CONCLUSIONS: Our results suggest the importance of evaluating DSAs against HLA-DP and -DQ in recipients before selecting CB units.

Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial.

Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.

Passive immune basophil activation test for the identification of allergic episodes from various adverse events elicited by haematopoietic cell transplantation: A pilot study.

BACKGROUND AND OBJECTIVES: Haematopoietic cell transplantation (HCT) therapy tends to be associated with various complications including engraftment failure, regimen-related toxicities, and infectious diseases. In addition, HC infusion itself occasionally elicits adverse events (AEs), one of the most common AEs is an allergic reaction. As appropriate laboratory tests have not yet been established to distinguish allergy-mediated AEs from other complications, clinical responses for HCT-related AEs can only be nonspecific. In this pilot study, using passive immune basophil activation test (pi-BAT), we attempted to distinguish an HC infusion-induced allergic reaction from various HCT-related AEs. MATERIALS AND METHODS: Using pi-BAT, we examined 34 patients who underwent HCT, that is, 11 with AEs and 23 without AEs as controls. RESULTS: Two of the eleven AE cases were pi-BAT positive and, the rest of nine AE cases were negative, while all non-AE cases were negative. Both of the two positive cases showed erythema, tachycardia, plus cough. Because erythema is one of the representative symptom of allergy, those cases could be classified as allergic reaction cases or anaphylaxis cases if tachycardia and cough were concomitant symptoms of erythema. Among the nine AEs with pi-BAT negative result, four cases showed urticaria, four showed vomiting plus diarrhoea, and one showed cough. Urticaria case was strongly suspected of allergy, however, the AE cases were pi-BAT negative. CONCLUSION: The pi-BAT may be useful as an auxiliary diagnostic tool to confirm the possible involvement of HC infusion in HCT-related AEs and identify an immunologic mechanism for HCT-related hypersensitivity reactions.

Sensitivity and specificity of passive immune-basophil activation test to detect allergic transfusion reactions.

BACKGROUND: The basophil activation test (BAT), performed with patient blood samples and supernatants from transfused blood, was developed to elucidate the mechanistic relationship between transfusion and the resultant allergic transfusion reactions (ATRs). This test cannot be performed on myelosuppressed patients and neonates because of the absence of basophils. Therefore, we devised the passive immune basophil activation test (pi-BAT) using patients' plasma and residual transfused blood as sources of immunoglobulin E and allergen, respectively, and the basophils of healthy volunteers served as a source of the responder cells. The sensitivity and specificity of the pi-BAT, however, remained largely unknown. STUDY DESIGN AND METHODS: In this study, the pi-BAT was performed on 31 patients with nonhemolytic transfusion reactions including nine non-ATR and 22 ATR (12 mild and 10 moderate-to-severe) cases to examine its sensitivity and specificity. RESULTS: Nine of the 10 cases with moderate-to-severe ATR tested positive, whereas all the non-ATR cases negative, strongly indicating immunoglobulin E and allergens are involved in the pathogenesis underlying the blood transfusion-triggered adverse effects. CONCLUSION: Thus, we propose that pi-BAT can be used to detect moderate-to-severe ATRs and their underlying mechanisms.

Immunoglobulin (Ig)G antibodies against IgE identified by basophil activation test as the putative causative agent of a serious allergic transfusion reaction: potential utility of the test as a new safety measure for allergic transfusion reactions.

BACKGROUND: In most cases of allergic transfusion reactions (ATRs), the causative agents have not been identified and the mechanisms are largely unknown, with a few exceptions. The basophil activation test (BAT) was recently introduced in the field of transfusion to investigate the causal relationships between ATRs and transfusion, as well as the mechanisms behind them. STUDY DESIGN AND METHODS: The BAT was used to screen the residual supernatants (SNs) of 43 blood components associated with serious ATRs for those that can activate basophils of many healthy volunteers. The SNs were then fractionated by centrifugal ultrafiltration and protein G column chromatography and each separated fraction was reexamined by the BAT. RESULTS: Of the 43 such blood components, one activated basophils from 19 of 21 healthy volunteers. In the blood component, the IgG antibody against IgE was identified as a putative causative agent. CONCLUSION: Blood donors who possessed the IgG antibody against IgE may be dangerous to transfusion recipients. The BAT would be useful in identifying such high-risk blood donors, when it is used to screen the blood components associated with serious ATRs for residual SNs that can activate the basophils of many healthy volunteers.

Possible Utility of the Basophil Activation Test for the Analysis of Mechanisms Involved in Allergic Transfusion Reactions.

Allergic transfusion reactions (ATRs) are the most common adverse reactions occurring during transfusion of blood components. Although most reactions are mild and involve cutaneous manifestations, severe ATRs including life-threatening anaphylaxis may also occur. The mechanisms of ATRs are largely unknown because they have not been well studied. One of the reasons for this may be the absence of a standard assay system for investigating these processes. Basophils and/or mast cells are key effector cells in immediate-type allergic reactions. They possess the unique ability to degranulate upon cross-linking of specific IgE bound on the membrane-bound, high-affinity IgE receptor or upon direct stimulation by exposure to allergens. Basophils are present in peripheral blood, unlike mast cells which are located in tissues. Therefore, basophils are valuable for the clinical testing of allergy. Consequently, the basophil activation test (BAT) was developed as a simple blood test for the diagnosis of allergic reactions to substances such as foods, inhalants, medicines and venom. In the last decade, the BAT has also been applied to transfusion medicine; 5 pilot studies revealed that the supernatants of the responsible blood products activated basophils in the BAT in 13 ATR cases, suggesting a causal relationship between ATRs and transfusion. In this review, we describe those cases and explore the potential utility of the BAT as a test performed in reference laboratories for the analysis of ATRs. We also describe the weaknesses, pitfalls, and unanswered issues of this assay.

Pulmonary Involvement of Acute Myeloid Leukemia Mimicking Transfusion-related Acute Lung Injury.

Transfusion-related acute lung injury (TRALI) is defined as a new episode of acute lung injury (ALI) occurring during transfusion or within 6 hours of transfusion completion. A 66-year-old man suffering from acute myeloid leukemia developed acute respiratory distress syndrome after platelet transfusion. TRALI was diagnosed clinically, but an autopsy showed leukemic cells in diffuse pulmonary edema. Anti-human neutrophil antigen (HNA)-3a antibodies were detected in the donor serum, and the HNA-3 genotype of the patient was identified as a/a. This case was considered to represent pulmonary involvement of acute myeloid leukemia, rather than TRALI. A revision of the definition of TRALI accounting for hematological malignancies should therefore be considered.

Clinical utility of a passive immune basophil activation test for the analysis of allergic transfusion reactions.

BACKGROUND: In previous studies, we demonstrated that the basophil activation test, which is performed using patient blood and the supernatants from transfused blood components, was able to elucidate not only the causative relationship between allergic transfusion reactions and the transfusion but also the mechanisms behind allergic transfusion reactions. However, for a large number of allergic transfusion reactions, patients are in a state of myelosuppression, and the basophil activation test cannot be performed for these patients because there are insufficient numbers of peripheral blood basophils. STUDY DESIGN AND METHODS: To overcome this obstacle, we developed a passive immune basophil activation test, in which patient plasma and residually transfused blood are used as the patient's sources of immunoglobulin E and allergen, respectively, whereas healthy volunteer basophils serve as the responder cell source. The passive immune basophil activation test was performed for two patients who had severe allergic transfusion reactions, using supernatants of the residual platelet concentrates and the patients' own immunoglobulin E. RESULTS: There were no differences in either surface immunoglobulin E or activation in response to allergens between untreated basophils and so-called quasi-basophils, in which immunoglobulin E was replaced by a third party's immunoglobulin E. In these patients, the supernatants of the residual platelet concentrates exclusively activated basophils in response to quasi-basophils onto which the patients' immunoglobulin E, but not a third party's immunoglobulin E, was bound. CONCLUSION: The passive immune basophil activation test may help clarify the causal relationship between allergic transfusion reactions and transfused blood, even when patients experience myelosuppression.

Mitochondrial damage-associated molecular patterns as potential proinflammatory mediators in post-platelet transfusion adverse effects.

BACKGROUND: Platelet concentrates (PCs) are the most common blood components eliciting nonhemolytic transfusion reactions (NHTRs), such as allergic transfusion reactions and febrile reactions. However, the precise mechanisms of NHTRs in PC transfusion remain largely unknown. Previous studies reported that mitochondria-derived damage-associated molecular patterns (DAMPs) could be important mediators of innate cell inflammation. Platelets (PLTs) represent a major reservoir of mitochondria in the blood circulation. The aim of this study was to determine the possible involvement of mitochondrial DAMPs in NHTRs. STUDY DESIGN AND METHODS: The amount of mitochondrial DAMPs was determined as an index of total copy numbers of mitochondrial DNA (mtDNA), including mtDNA itself and free mitochondria, using quantitative real-time polymerase chain reaction. To examine whether neutrophils, monocytes, and basophils were activated by mitochondrial DAMPs in vitro, an in vitro whole blood cell culture assay was performed. RESULTS: In blood components associated with NHTRs, the mean total mtDNA concentration was highest in PCs followed in order by fresh-frozen plasma and red blood cells. The amount of mtDNA in NHTR PCs was higher than that in control PCs without NHTRs. The mitochondrial DAMPs present in NHTR PCs was high enough to activate neutrophils, monocytes, and basophils, when costimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or HLA antibodies. CONCLUSION: PLT-derived mitochondrial DAMPs are candidate risk factors for the onset of NHTRs.

High-resolution melting analysis as an alternative method for human neutrophil antigen genotyping.

Human neutrophil antigen (HNA)-typed granulocyte panels are widely used to screen for the presence of HNA antibodies and to determine antibody specificity. Many laboratories screen donors for HNA genotypes using low-throughput methods such as allele-specific polymerase chain reaction (PCR), PCR-restriction fragment-length polymorphism, and multiplex PCR. In the present study, we used a high-resolution melting (HRM) analysis to determine HNA genotypes. For the HRM analysis, purified genomic DNA samples were amplified via PCR with HNA-specific primers. Nucleotide substitutions in genes encoding HNAs were differentiated on the basis of the HRM curves, and the results of HRM and DNA sequencing analyses were determined to be in complete agreement. The gene frequency of HNA-1a, -1b, -1c, -3a, -3b, -4a, -4b, -5a, and -5b in the Japanese population was consistent with the previous reports. Our results suggest that HRM analysis can be used for genotyping HNA antigens determined by single nucleotide substitutions.

Proposed criterion for distinguishing ABO mosaics from ABO chimeras using flow cytometric analysis.

Differentiation of ABO mosaics from chimeras is performed using flow cytometry (FCM) analysis. Although mosaics and chimeras have been distinguished by presence or absence of clear resolution using FCM analysis, the lack of quantitative metrics and definitive criteria for this differentiation has made some cases difficult to differentiate. In this study, therefore, we attempted to establish a definitive and quantitative criterion for this differentiation. When FCM histogram gates for group "A" or "B" antigen-negative and -positive red blood cells (RBCs) were set such that group O RBCs were classified as 99 percent negative and group A or B RBCs as 99 percent positive, the percentages of RBCs in the middle region of six chimeras and 23 mosaics (12 A mosaics and 11 B mosaics) were 0.1-0.6 percent and 7.0-19.0 percent, respectively. This results suggested that ABO mosaics and chimeras can be unambiguously differentiated when the cutoff point of the intermediate region is set to 1 percent.

The IgE-dependent pathway in allergic transfusion reactions: involvement of donor blood allergens other than plasma proteins.

On transfusion, several plasma proteins can cause anaphylaxis in patients deficient in the corresponding plasma proteins. However, little is known about other allergens, which are encountered much more infrequently. Although it has been speculated that an allergen-independent pathway underlying allergic transfusion reactions (ATRs) is elicited by biological response modifiers accumulated in blood components during storage, the exact mechanisms remain unresolved. Furthermore, it is difficult even to determine whether ATRs are induced via allergen-dependent or allergen-independent pathways. To distinguish these two pathways in ATR cases, we established a basophil activation test, in which the basophil-activating ability of supernatants of residual transfused blood of ATR cases to whole blood basophils was assessed in the presence or absence of dasatinib, an inhibitor of IgE-mediated basophil activation. Three of 37 supernatants from the platelet concentrates with ATRs activated panel blood basophils in the absence, but not in the presence, of dasatinib. The basophil activation was inhibited by treatment of anti-fish collagen I MoAb in one case, suggesting that the involvement of fish allergens may have been present in donor plasma. We concluded that unknown non-plasma proteins, some of which had epitopes similar to fish antigens, in blood component may be involved in ATRs via an allergen/IgE-dependent pathway.

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies.

Granulocyte immunofluorescence and granulocyte agglutination tests are standard methods for detecting human neutrophil antigen (HNA) antibodies (Abs); however, these require a typed panel of neutrophils, which can be time-consuming to develop, and it remains difficult to determine antibody specificity in some cases. We established and evaluated four detection systems for HNA-1a Abs based on an HNA-1a-expressing cell line (KY cells) and antigen capture. We additionally evaluated a commercial solid-phase system. Eleven HNA-1a antibody-positive samples, including the World Health Organization Reference Reagent, and 40 serum samples derived from male blood donors were used as positive and negative control samples, respectively. Although specificity was >0.90 in all systems evaluated, the sensitivity varied among the systems. The KY cell-based monoclonal antibody specific immobilisation of granulocyte antigens (KY-MAIGA) system using certain, but not all, monoclonal Abs, and the solid-phase system revealed higher sensitivity than other systems. In conclusion, the KY-MAIGA and commercial solid-phase systems were superior in terms of specific and sensitive detection of HNA-1a Abs.

Detection of anti-human platelet antibodies against integrin α2ß1 using cell lines.

BACKGROUND: Antibodies against human platelet antigens (HPA) are a cause of thrombocytopenia. Detection of rare anti-HPA antibodies using platelet preparations is difficult and would be improved by an alternative method that does not require platelets. In the present study, we describe the establishment of cell lines that stably express specific HPA associated with integrin α2ß1 and the application of these cell lines for detecting anti-HPA-5a and anti-HPA-5b antibodies. MATERIALS AND METHODS: Complementary DNA of the integrin α2 variants HPA-5b, -13b and -18b were individually transfected into K562 cells using retroviral vectors. Expression of integrin α2 was confirmed by flow cytometric analysis, immunoprecipitation and western blotting analysis. To verify whether the cell line panel was suitable for clinical diagnosis, we analysed its properties using monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) and well-characterised serum samples. RESULTS: Exogenous integrin α2 expression was observed in the transfected cells for over 6 months. The cell line panel specifically detected previously characterised anti-HPA-5a and anti-HPA-5b antisera. No reactivity was observed with control sera, including normal sera and HLA antisera. DISCUSSION: We successfully established a cell line panel to facilitate the sensitive and reliable detection of anti-HPA-5a and anti-HPA-5b antibodies.

The first two cases of neonatal alloimmune thrombocytopenia associated with the low-frequency platelet antigen HPA-21bw (Nos) in Japan.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a disorder characterized by maternal alloimmunization against paternal fetal platelet antigens. Two healthy, unrelated Japanese women each gave birth to a child with severe NAIT. STUDY DESIGN AND METHODS: To elucidate the maternal causes of NAIT, we conducted serologic and genetic studies in these two NAIT infants. RESULTS: The serologic experiments localized the antigens to the glycoprotein (GP) IIIa subunit of the GPIIb/IIIa complex. Sequence-based typing studies subsequently identified a G>A mutation at Nucleotide 1960 (a glutamic acid > lysine substitution at Position 628) in the 11th exon of the GPIIIa gene. This mutation was recently identified in a report as HPA-21bw. Next, it was determined that the cause of NAIT in both cases was the HPA-21bw antigen, as shown by the mothers' antibodies reacting with the mutated GPIIIa-transfected cells, but not with transfectants expressing wild-type GPIIIa. A molecular genetic screening for the HPA-21bw allele among Japanese donors showed that its genetic frequency in the population was 0.53% (10/1888), indicating that HPA-21bw occurs at a low but appreciable frequency in the population. Furthermore, in a retrospective study of 50 previous NAIT cases of unknown causes, we found one NAIT case associated with the HPA-21bw antibody. The two NAIT cases in this study represent the first ones to be associated with HPA-21bw in Japan. CONCLUSION: We identified the HPA-21bw allele from two unrelated Japanese infants with severe NAIT. We identified 10 individuals (1.06%) positive for the HPA-21bw allele from a genetic screening of 944 Japanese blood donors.