A novel swab storage gel is superior to dry swab DNA collection, and enables long-range high resolution next generation sequencing HLA typing from buccal cell samples.

HLA sequence-based DNA typing (SBT) by long-range PCR amplification (LR PCR) and next-generation sequencing (NGS) is a high-throughput DNA sequencing method (LR-NGS-SBT) for the efficient and sensitive detection of novel and null HLA alleles to the field-4 level of allelic resolution without phase ambiguity. However, the accuracy and reliability of the HLA typing results using buccal cells (BCs) and saliva as genetic source materials for the LR-NGS-SBT method are dependent largely on the quality of the extracted genomic DNA (gDNA) because a large degree of gDNA fragmentation can result in insufficient PCR amplification with the incorrect assignment of HLA alleles because of allele dropouts. In this study, we developed a new cost-efficient swab storage gel (SSG) for wet swab collection of BCs (BC-SSG) and evaluated its usefulness by performing different DNA analytical parameters including LR-NGS-SBT to compare the quality and quantity of gDNA extracted from BCs (in SSG or air dried), blood and saliva of 30 subjects. The BC-SSG samples after 5 days of storage revealed qualitative and quantitative DNA values equivalent to that of blood and/or saliva and better than swabs that were only air-dried (BC-nSSG). Moreover, all the gDNA extracted from blood, saliva and BC-SSG samples were HLA-typed successfully to an equivalent total of 408 alleles for each sample type. Therefore, the application of BC-SSG collection media for LR-NGS-SBT has benefits over BC dried samples (dry swabs) such as reducing retesting and the number of untestable BC samples because of insufficient DNA amplification.

A case of Philadelphia chromosome-positive acute lymphocytic leukaemia with type I CD36 deficiency.

BACKGROUND: CD36 is a glycoprotein expressed on platelets and monocytes of the blood. There are two types of CD36 deficiency, type I and type II. Individuals with type I-deficiency do not express CD36 in any cell type and can produce the CD36 antibody, which causes pathological conditions, such as fetal/neonatal alloimmune thrombocytopenia (FNAIT) and platelet transfusion refractory (PTR), through antigenic exposure via transfusion or pregnancy. CASE PRESENTATION: We experienced a case of Philadelphia-positive acute lymphoblastic leukaemia with PTR. In addition to the CD36 antibody, multiple-specificity HLA antibodies were present in the patient's plasma, requiring transfusion of HLA-compatible and CD36-negative platelets (PC-HLA). Since the number of donors was limited, it was necessary to set-up a blood transfusion schedule so that hyper-fractionated cyclophosphamide, vincristine and doxorubicin therapy (hyper-CVAD) and ponatinib combination chemotherapy could be safely administered to achieve molecular remission. Rituximab administration resulted in reduced levels of both CD36 antibody and HLA antibody. Given the expression of CD36 on haematopoietic stem cells and the limited availability of CD36-negative PC-HLA, haematopoietic stem cell transplantation (HSCT) was not considered to be an option. CONCLUSION: If CD36-negative, allogeneic haematopoietic stem cell donors are unable to be found, the indications for HSCT in patients with type I CD36-deficiency should be carefully weighed. In the present case, molecular remission has been able to be maintained to the present day after completion of a two-year maintenance regimen.

Does time of CCI measurement affect the evaluation of platelet transfusion effectiveness?

The measurement of corrected count increment at 1-h post-transfusion (CCI-1 h) of platelet concentrate (PC) transfusion is recommended, but in the revised Japanese Guideline (2017) it was changed to "after 10-min to 1-h", following the revision of the guidelines from Western countries. Here, we aimed to investigate on the feasibility to apply the CCI measured at 10-min or 30-min post-transfusion as the surrogate of CCI-1 h. Peripheral blood was collected at 10-min, 30-min and 1-h post-transfusion of PC and the effectiveness of the transfusion was analyzed based on the CCI. In the period from December 2017 to February 2020, 8 patients, who received multiple PC transfusion (total 208) at our institution, were analyzed. We performed the univariate analyses to examine the relationship between CCI value and the categorical variables, p-value <0.1 was obtained for gender (p = 2.91 × 10-19), fever after transfusion (p = 0.0163). The qualitative variables, namely measurement time (p = 0.0553), also showed p-value <0.1. Using these factors as covariates in the mixed effect model, we found that the measurement time (p = 0.0007) had a significant effect on the CCI value when looking at fixed effects. Although there is a tendency for decreased CCI values with time progression, the slope of the change in the mixed model was -0.00307, indicating that the CCI difference among the 3 measurements was small. Here we provide evidence that CCI measured at 10-min and 30-min post-transfusion give results comparable to those measured at 1-h post-transfusion, under the Japanese practice of platelet transfusion, which relies on 100 % single-donor apheresis PC, and ABO-identical whenever possible.

Marked thrombocytopenia in a neonate is associated with anti-HPA-5b, anti-HLA-A31, and anti-HLA-B55 antibodies.

Maternal antibodies against human platelet antigen (HPA) and/or human leukocyte antigen (HLA) cause fetal and neonatal alloimmune thrombocytopenia (FNAIT) in 0.09-0.15% of live births. Severe cases account for 5-31% and the frequency of multiple kinds of alloantibodies is 6.9-9% of FNAIT. We present a case of severe FNAIT associated with anti-HPA-5b, anti-HLA-A31, and anti-HLA-B55 antibodies, successfully treated with immunoglobulin and platelet transfusion. The anti-HLA-B55 antibody was detected in the newborn's serum, but disappeared on the 20th day, which was followed by an increase of the platelet count. These findings suggested the potential involvement of an anti-HLA antibody in the pathogenesis of FNAIT.

Human primary biliary cirrhosis-susceptible allele of rs4979462 enhances TNFSF15 expression by binding NF-1.

A genome-wide association study (GWAS) identified tumor necrosis factor superfamily member 15 (TNFSF15) as the strongest associated gene with susceptibility to primary biliary cirrhosis (PBC) outside the HLA loci in the Japanese population. However, causal functional variants of the TNFSF15 locus and the molecular mechanism underlying disease susceptibility have not been clarified. Here, to identify the functional causal variants of the TNFSF15 locus, integrated analysis comprising in silico analysis, a case-control association study and in vitro functional analysis was performed. Initially, 32 functional candidate single-nucleotide polymorphisms (SNPs) in the expression regulatory motifs, the coding region, or the untranslated regions (UTRs) of the TNFSF15 locus were selected by in silico analysis. By the case-control association studies using PBC patients (n = 1279) and healthy controls (n = 1091) in the Japanese population, rs4979462 [P = 1.85 × 10(-14) (our previous study)], rs56211063 (P = 2.21 × 10(-14)), and rs55768522 (r(2) = 1 with rs4979462) were likely candidates for causal variants. Among these SNPs, rs4979462 was identified as the causal variant by in vitro functional analysis using luciferase assay and electrophoretic mobility shift assay (EMSA). Super-shift assay clarified that PBC-susceptible allele of rs4979462 generated a novel NF-1 binding site. Moreover, higher endogenous TNFSF15 protein and mRNA expression levels were observed in individuals with the PBC-susceptible allele of rs4979462. This study identified the causal variant for PBC susceptibility in the TNFSF15 locus and clarified its underlying molecular mechanism. TNFSF15 and NF-1 are considered to be potential targets for the treatment of PBC.

Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness.

BACKGROUND: Multiple platelet exposure induces anti-HLA and/or anti-HPA antibody production, which may cause platelet transfusion refractoriness (PTR). In Japan, the universal pre-storage leukocyte reduction (ULR) was fully implemented since 2006, but prior to ULR, in our institution, leukocyte reduction filters were routinely used at the bedside (bedside leukoreduction, BSLR) for all onco-hematological patients receiving multiple platelet transfusions. OBJECTIVE: We retrospectively compared patients receiving platelet transfusions in the era of ULR with those of BSLR era. MATERIALS AND METHODS: Patients of the BSLR group (409 cases) and the ULR group (586 cases) were compared in terms of alloimmunization and immunological PTR. The clinico-pathological features, including gender, history of pregnancy, number of exposed transfusion donors, periods of transfusion, and prior stem cell transplantation were compared, and the risk factors of alloimmunization were determined. RESULTS: The antibody detection rate was significantly higher in the ULR compared to BSLR group (8.7% vs. 5.4%), as well as the immunological PTR rate (7.3% vs. 3.2%). By the multivariate analysis, female gender and the number of platelet donor exposure, but not universal leukoreduction or transfusion period, were found to be the risk factors strongly associated with alloantibody formation. CONCLUSION: Although ULR may be superior to BSLR in terms of preventing non-hemolytic transfusion reactions, BSLR was found to be as effective as ULR in terms of preventing platelet alloimmunization and refractoriness. Thus, BSLR should be actively indicated as a realistic alternative in developing countries, before the universal leukoreduction is fully implemented.

Novel swine model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available. STUDY DESIGN AND METHODS: To develop a swine model of TRALI, male Clawn strain miniature pigs were used. A monoclonal antibody (MoAb) against swine leukocyte antigens (SLAs) Class I (4G8, 0.3 or 1.0 mg/kg body weight [BW]) and a control antibody (1.0 mg/kg BW) were injected into the peripheral vein after priming with or without 1 µg/kg BW lipopolysaccharide (LPS; n = 3 each). Lung injury was assessed using PaO2 /FiO2 (P/F) ratio and by chest X-ray imaging. Histopathologic analysis was also conducted. RESULTS: Lung injury could be induced by injecting 4G8 at an amount of 1.0 mg/kg BW, after LPS. The P/F ratio 90 minutes after the administration of 4G8 significantly decreased (p < 0.05). Bilateral infiltration was shown in chest X-ray imaging. Lung injury was confirmed by histopathologic analysis. CONCLUSION: Lung injury in pigs was successfully induced by anti-SLA MoAb. Priming with LPS is a prerequisite for inducing lung injury and the amount of the antibody is a critical condition.

The role of alloantibodies against human platelet antigen-15 in multiply platelet transfused patients.

BACKGROUND: Several studies have documented the role of antibodies against human platelet (PLT) antigen (HPA)-15 in alloimmune-mediated thrombocytopenia including neonatal alloimmune thrombocytopenia, PLT transfusion refractoriness (PTR), and posttransfusion purpura in Caucasian persons. However, the relevance of anti-HPA-15 in PTR among the Japanese population is still unclear. STUDY DESIGN AND METHODS: The sera of 305 multiply PLT transfused (MPT) patients, previously investigated for the presence of human leukocyte antigen (HLA) and HPA antibodies by mixed passive hemagglutination, were reexamined for the presence of HPA-15 alloantibodies, using the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) technique. RESULTS: Among the 305 MPT samples, antibodies against HPA-15 alloantigen was detected in seven (2.3%), two (0.66%) being anti-HPA-15a and five (1.64%) being anti-HPA-15b. Additionally, one case of CD109 panreactive antibody was found (0.33%). Among them, one aplastic anemia patient with blood group O developed multispecific anti-HLA and anti-HPA-15b alloantibody after MPTs. However, transfusion with HLA-matched PLTs of blood group AB did not result in adequate PLT count increment. Analysis of the possible influence of immune anti-A and anti-B by the MAIPA assay resulted negative, indicating that anti-HPA-15b is responsible for the refractory state in this patient. CONCLUSION: In this study, we found alloimmunization against HPA-15a and -15b in Japanese populations and demonstrated the relevance of these antibodies in a patient with PTR.

The current status of autologous blood transfusion in Japan--the importance of pre-deposit autologous blood donation program and the needs to achieve patient blood management.

BACKGROUND: Autologous blood transfusion (ABT) is currently considered the safest transfusion, since the risks of allogeneic immunological reaction and viral transmission are theoretically null. Although its use has declined in Western countries in the recent decade, it has been progressively expanded in Japan. With the widening of the concept of patient blood management (PBM), which aims to prevent the harmful adverse effects of the exposure to allogeneic blood, the importance of the ABT has once again gained interest. STUDY DESIGN AND METHODS: Here, we retrospectively analyzed the cases pre-depositing autologous blood for an elective surgery in the period of January 2000 to December 2010 in our hospital, where a pre-deposit autologous blood donation (PAD) program has been established in 2006, in an attempt to analyze the improvements achieved, and the problems remaining to achieve patient blood management. RESULTS: The PAD program contributed for the further improvement of ABT, and the number of participating patients increased, especially in the period 2002-2003, when the idea of PAD program implementation came out. By simple extrapolation of the ABT data to allogeneic blood, ABT was found to be superior in terms of cost-effectiveness. However, problems such as the high wastage rate, and the inappropriate transfusion triggers remain to be solved. CONCLUSION: ABT plays the central role in PBM, but to achieve the real PBM, there is need to indicate ABT appropriately, according to the individual needs, and use it adequately, without discarding. Our present data reflect the present status of the ABT performance in Japan, and will serve as the basis for the development of strategies to achieve safe and appropriate performance of ABT, and consequently, achieve PBM.

Inhibition of lysophosphatidic acid increase by prestorage whole blood leukoreduction in autologous CPDA-1 whole blood.

BACKGROUND: Lysophosphatidylcholine (LPC) has been implicated in the onset of transfusion-related acute lung injury (TRALI). In plasma, LPC is converted to lysophosphatidic acid (LPA) by autotaxin (ATX). The effect of leukoreduction in the accumulation of these bioactive lipids and ATX in human autologous blood has not been fully investigated. STUDY DESIGN AND METHODS: The accumulation of choline-containing phospholipids (LPC, sphingomyelin [SM], and phosphatidylcholine [PC]), LPA, and ATX during the storage of autologous blood and the changes caused by leukoreduction were investigated. A total of 26 orthopedic patients were enrolled. Autologous blood was collected as whole blood and, after leukoreduction, preserved refrigerated until use. Prestorage leukoreduced (LR) and non-LR autologous blood samples were analyzed. The time-dependent changes and the effect of the filtration were compared. RESULTS: A time-dependent and significant increase in the levels of LPA was observed in both non-LR and LR samples. The concentration of LPA was significantly reduced in LR compared to non-LR samples. The concentration of LPC was higher in LR compared to non-LR samples. The levels of PC, SM, and ATX were not affected by either the storage period or the leukoreduction. CONCLUSIONS: Leukoreduction of autologous whole blood effectively reduced the accumulation of LPA. On the other hand, prestorage leukoreduction resulted in an increased concentration of LPC, without significantly affecting ATX. Further studies are necessary to confirm the role of LPA in the pathogenesis of adverse effects of blood transfusion, especially TRALI.

The effect of pre-storage whole-blood leukocyte reduction on cytokines/chemokines levels in autologous CPDA-1 whole blood.

BACKGROUND: In this study, we aimed to investigate the effectiveness of pre-storage leukocyte filtration of autologous blood (AB), especially focusing on the cytokines/chemokines accumulation on blood products. MATERIALS AND METHODS: After approval of the ethics committee of the University of Tokyo, a total of 26 orthopedic patients, who donated AB prior to surgery after informed consent, were enrolled. The effects of filtration on blood cell counts were analyzed, and the accumulation of cytokines and chemokines were measured on pre- and post-leukoreduced (LR) samples, using the Luminex system. The time-dependent changes of the cytokines/chemokines and the effect of the filtration on their concentration were analyzed, and compared with the normal plasma levels reported in the literature. RESULTS: LR effectively reduced the number of leukocytes and platelets, without affecting that of red cells. The concentration of most of the cytokines/chemokines analyzed, except the EGF, sCD40-L and sFas-L, decreased time-dependently of storage or did not change in pre-LR samples. However, EGF, sCD40L and sFas-L were significantly reduced by LR. Some, such as IL-8 and RANTES, were also importantly decreased by LR, and others, such as IL-1ß and TNF-α, were not significantly affected by LR. CONCLUSIONS: Leukocyte filtration effectively removes platelets and leukocytes from AB, thus preventing the accumulation of cytokines/chemokines. Since adverse effects due to AB transfusion, although rare, are observed, there is need to consider the implementation of pre-storage leukocyte reduction (PSLR) for AB.

The first case of alloantibody against human platelet antigen-15b in Japan: possible alloimmunization by a hydatidiform mole.

BACKGROUND: The involvement of the human platelet antigen (HPA)-15 system in neonatal alloimmune thrombocytopenia (NAIT) has been reported in various populations, but not in the Japanese population. In Japan, the mixed passive hemagglutination assay (MPHA) is used for detection of HPA alloantibodies. However, most of the reported cases of HPA-15 incompatibility are based on the monoclonal antibody immobilization of platelet antigen (MAIPA) assay or immunoprecipitation; thus there is a possibility that HPA-15 alloantibodies are not efficiently detected by the MPHA, and currently, the causative antibody is not detectable in approximately half of the suspected NAIT cases in Japan. STUDY DESIGN AND METHODS: We examined the sera of mothers from NAIT cases, previously with undetected HPA antibodies by MPHA, using the MAIPA technique. Sera from 90 mothers of suspected NAIT were tested by MAIPA for the presence of anti-HPA-15 alloantibodies. RESULTS: Anti-HPA-15b was detected in one case. This case was a mother in the first pregnancy diagnosed as hydatid mole-coexisting fetus, and the baby was born with suspected NAIT. The familial analysis revealed compatibility of HPA-15 genotype between the mother and the baby (both HPA-15a/a), but incompatibility with the paternal one (HPA-15a/b). The hydatid mole's tissue was genotyped as HPA-15b positive. Besides anti-HPA-15b, maternal sera contain strong HLA Class I antibody CONCLUSIONS: Here we reported the first case of anti-HPA-15 in Japan. Alloimmunization against the hydatid mole seems to be responsible for the production of HPA-15b alloantibody. This antibody, however, did not apparently involve in the development of NAIT of the newborn, the coexisting anti-HLA Class I being the possible cause.

Pilot study of anti-angiogenic vaccine using fixed whole endothelium in patients with progressive malignancy after failure of conventional therapy.

Vaccines targeting tumour angiogenesis were recently shown to inhibit tumour growth in animal models. However, there is still a lack of information about the clinical utility of anti-angiogenic vaccination. Therefore, here, we aimed to test the clinical effects of a vaccine using glutaraldehyde-fixed human umbilical vein endothelial cells (HUVECs). Six patients with recurrent malignant brain tumours and three patients with metastatic colorectal cancer received intradermal injections of 5x10(7) HUVECs/dose (in total 230 vaccinations). ELISA and flow cytometry revealed immunoglobulin response against HUVECs' membrane antigens. ELISPOT and chromium-release cytotoxicity assay revealed a specific cellular immune response against HUVECs, which were lysed in an effectors:targets ratio-dependent manner. Gadolinium-contrasted MRI showed partial or complete tumour responses in three malignant brain tumour patients. Except for a DTH-like skin reaction at the injection site, no adverse effect of vaccination could be observed. Our results suggest that the endothelial vaccine can overcome peripheral tolerance of self-angiogenic antigens in clinical settings, and therefore should be useful for adjuvant immunotherapy of cancer.

CD11b-mediated migratory property of peripheral blood B cells.

BACKGROUND: CD11b belongs to the integrin family and is expressed on neutrophils, monocytes, natural killer cells, and a subset of lymphocytes. Although CD11b expressed on neutrophils and monocytes has been extensively investigated and has been reported to play an important role in the migration of these subsets of leukocytes, the function of CD11b expressed on a subset of B cells has not yet been clarified. OBJECTIVE: To elucidate the functional activity of CD11b expressed on B cells, we characterized the CD11b-expressing cells among the B-cell population and investigated their migratory ability. METHODS: Isolated peripheral blood CD19 + B cells were analyzed by flow cytometry. The migratory ability of B cells was evaluated by the transwell assay, and the contribution of CD11b to this ability was investigated by using an anti-CD11b blocking mAb. RESULTS: The majority of CD27 - IgD + naive B cells were CD11b - , whereas most CD27 + memory cells were CD11b +. Among the CD27 + memory cells, expression of CD11b was stronger on the IgD - cells than on the IgD + cells. In the transwell assay, the migrating cells were predominantly CD27 + IgD - cells, most of which expressed CD11b. The addition of an anti-CD11b blocking mAb resulted in the significant reduction of the number of migrating B cells. CONCLUSION: Memory B cells express CD11b and, in contrast with naive B cells, have high migratory ability. CD11b plays an essential role in the homing process of memory cells.