A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)-1a and -1b antibodies.

BACKGROUND: To identify specific human neutrophil antigen (HNA) antibodies, assays using neutrophils such as monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) are recommended. However, these assays are limited by labor-intensive neutrophil preparation and varying antigen expression levels. METHODS: We evaluated a newly developed immunocomplex capture fluorescence assay (ICFA) for identifying HNA-1 antibodies and compared it to MAIGA and LABScreen Multi (LABM), which utilizes recombinant HNA-coated Luminex beads. For ICFA, HNA-1a or HNA-1b transfected cells replaced neutrophils. Cells incubated with serum were lysed, and immune complexes were captured using five CD16 monoclonal antibody-conjugated Luminex beads. Nine antisera with known specificity and 26 samples suspected of containing HNA antibodies were analyzed by ICFA and MAIGA using neutrophils or transfected cells (ICFA-N or ICFA-T, and MAIGA-N or MAIGA-T, respectively). RESULTS: ICFA-T and MAIGA-N accurately determined the specificity of all antibodies in the nine antiserum samples. The ICFA-T detection limit was 2048-fold for anti-HNA-1a and 256-fold for anti-HNA-1b; the limits of MAIGA-T, MAIGA-N, and LABM were 32-, 4 ~ 64-, and 128-fold for anti-HNA-1a and 64-, 16 ~ 64-, and 32-fold for anti-HNA-1b, respectively. Twelve and 7 of the remaining 26 samples tested negative and positive, respectively, in both ICFA-T and MAIGA-N. Antibody specificity against HNA-1a or HNA-1b determined using ICFA-T agreed with that determined using MAIGA-N and LABM. Another seven samples tested positive in ICFA-T but negative in MAIGA-N. CONCLUSION: The novel ICFA is highly sensitive and exhibits specificity similar to MAIGA and LABM for detecting HNA-1 antibodies.

Overlapping ADAMTS13 peptide binding profiles of DRB1∗08:03 and DRB1∗11:01 suggest a common etiology of immune-mediated thrombotic thrombocytopenic purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare autoimmune disorder caused by autoantibodies against ADAMTS13. A strong association of DRB1∗11 with iTTP and DRB1∗11-restricted T-cell epitopes in ADAMTS13 have been reported in Europeans, whereas we previously found DRB1∗08:03 as a susceptible allele in Japanese. OBJECTIVES: The limited information is available regarding a susceptible allele and its T-cell epitopes in Japanese patients with iTTP. MATERIALS AND METHODS: We conducted a reanalysis on iTTP-predisposing alleles using 3 distinct Japanese control groups. Subsequently, a novel human leukocyte antigen (HLA)-peptide expression assay (MHC-density assay) was used to identify the presentation of 24 ADAMTS13-derived peptides, including the regions that were identified previously by MHC-peptidome analysis and/or T-cell assays or predicted by NetMHCIIpan-4.0, to DRB1∗08:03 and DRB1∗11:01. RESULTS: We reconfirmed the strong association of DRB1∗08:03 with iTTP, as well as the absence of the secondary risk alleles and protective alleles in Japanese iTTP, which altogether reveal that the HLA association pattern is completely different between the European and Japanese iTTP. MHC-density assay found the 3 ADAMTS13-derived peptides in the spacer domain as a potential strong binder to DRB1∗08:03. Moreover, 6 peptides in the metalloprotease, spacer, sixth thrombospondin-1 repeat, and CUB domains in ADAMTS13 showed increased presentation by both DRB1∗08:03 and DRB1∗11:01. CONCLUSION: Altogether, the findings of distinct HLA-DR association with iTTP across populations and the presentation of common peptides by DRB1∗08:03 and DRB1∗11:01 suggest that the same ADAMTS13-derived peptides might be presented and trigger the activation of autoreactive CD4+ T cells, leading to production of anti-ADAMTS13 autoantibodies by autoreactive B cells.

Association of HLA-DRB1*15:02:01, DQB1*05:01:24 and DPB1*13:01:01 in Thai patients with systemic sclerosis.

HLA studies in patients with systemic sclerosis (SSc) have shown variable results. This study aimed to examine the association of HLA class I and II risk alleles in Thai SSc patients, and clarify the contribution of risk HLA alleles to the pathogenesis and clinical manifestations. Blood samples from 92 SSc patients and 135 healthy controls (HCs) were collected. Eleven loci of the HLA class I (HLA-A, B, and C) and class II (HLA-DR, DP, and DQ) genes were determined by a 3-field (6-digit) analysis using the Next Generation DNA Sequencing (NGS) method. Anti-topoisomerase-I antibodies (ATA) and anti-centromere antibodies (ACA) were identified by ELISA methods. Allele frequencies (AFs) of HLA-DRB1*15:02:01, DRB5*01:02:01, DQB1*05:01:24, DPB1*13:01:01, and DQA1*01:01:01 were increased significantly in the whole SSc and SSc patients with positive ATA, but with negative ACA (SSc/ATA+/ACA-). Of these, DPB1*13:01:01 was the most susceptible allele. The DRB1*15:02:01, DQB1:05:01:24, and DPB1*13:01:01 alleles were estimated to locate on the unique haplotype, and haplotype frequency was estimated to be significantly higher than those in the HCs (p = 0.002). The linkage analysis of DRB1*15/16 revealed that most of the DRB1*15:02:01 alleles were linked to DRB5*01:02:01 or DRB5*01:08:01N. The linkage of DRB1*16:02:01 to DRB5*01:01:01 was observed frequently. The associations of risk alleles with several SSc clinical features were observed. HLA-DRB1*15:02:01, DRB5*01:02:01, DQB1*05:01:24, and DPB1*13:01:01 on the unique haplotype were associated with the pathogenesis and clinical features of SSc in Thai patients. The linkage of DRB1*15:02:01 to DRB5*01:08:01N was observed commonly in northern Thai patients.

Cord blood index predicts engraftment and early non-relapse mortality in adult patients with single-unit cord blood transplantation.

How to select optimal cord blood (CB) remains an important clinical question. We developed and validated an index of CB engraftment, the cord blood index (CBI), which uses three weighted variables representing cell doses and HLA mismatches. We modeled the neutrophil engraftment time with competing events by random survival forests for competing risks as a function of the predictors: total nucleated cells, CD34, colony-forming units for granulocytes/macrophages, and the number of HLA mismatches at the antigen and allele levels. The CBI defined three groups that had different neutrophil engraftment rates at day 30 (High, 83.7% [95% CI, 79.2-88.1%]; Intermediate, 77.0% [95% CI, 73.7-80.2%]; Low, 68.4% [95% CI, 63.6-73.2%]), platelet engraftment rates at day 60 (High, 70.4% [95% CI, 64.9-75.9%]; Intermediate, 62.3% [95% CI, 58.5-66.0%]; Low, 49.3% [95% CI, 44.2-54.5%]), and non-relapse mortality at day 100 (High, 14.1% [95% CI, 9.9-18.3%]; Intermediate, 16.4% [95% CI, 13.5-19.3%]; Low, 21.3% [95% CI, 17.1-25.5%]). This novel approach is clinically beneficial and can be adopted immediately because it uses easily obtained pre-freeze data of CB.

Improved detection of donor-specific HLA-class II antibody in kidney transplant recipients by modified immunocomplex capture fluorescence analysis.

Immunocomplex capture fluorescence analysis (ICFA) which basic principle is same as Luminex crossmatch (LXM), could detect donor-specific HLA antibody (DSA). The advantages of ICFA are (i) detection of DSA and (ii) no requirement of viable cells over the flow cytometry crossmatch (FCXM). However, FCXM has been widely used because of its higher sensitivity than ICFA, in particular HLA-class II antibody detection. In this study the accuracy of DSA detection against HLA-class II was investigated by modifying the original method of ICFA. Increment of the sensitivity was found when purified peripheral blood mononuclear cells (PBMCs) were used instead of whole blood. An ICFA-PBMC in addition to FCXM-T/B was conducted for 118 patients before kidney transplantation and 13 patients with de novo DSA against HLA-class II after transplantation. Significantly positive correlation was observed between the values of ICFA-PBMC and DSA mean fluorescence intensity (MFI) targeting class II (p < 0.0001). When the cutoff level of 1.4 was determined by receiver operating characteristic curve analysis, the average DSA MFI was found to be significantly higher in the ICFA-PBMC (class II) positive group comparing to that in the negative group (12,217 vs 3885, p = 0.0027). ICFA-PBMC and optimized cutoff level could provide valid information in cases of suspected DSA.

Implications of HLA diversity among regions for bone marrow donor searches in Japan.

Japan is an island country, and the Japanese people have had minimal genetic exchange with other ethnolinguistic groups. Consequently, the population is highly uniform and has limited HLA diversity relative to people from other countries. However, Japan has three ethnolinguistic groups, and HLA distributions differ depending on geographic region. To collect an HLA-rich variety of bone marrow bank donor registrants, it is essential to know the precise distribution of HLA in Japan. We analyzed HLA alleles and haplotypes based on HLA information of 177 041 bone marrow donor registrants. Registrants were grouped depending on the prefecture and region (a group of prefectures) as commonly used in Japan. The prefectures did not show the same distributions, but the tendency was similar for each region. We found that Okinawa Prefecture and the mainland can be clearly divided as haplotypes: [A*24:02-C*01:02-B*54:01-DRB1*04:05] and [A*24:02-C*01:02-B*59:01-DRB1*04:05] were typically found in Okinawa (P = .02, P < .001). Moreover, these types were found almost exclusively in Japan and Korea. Donor registration centers of the Japan Marrow Donor Program are currently located in all prefectures. It is essential to deploy registration centers to collect registrants with a large variety of HLA types covering all of Japan.

A new HLA-C allele with an alternative splice site in exon 3: HLA-C*03:23N.

We identified a probable new null HLA-C allele, C*03:23N, which originated from C*03:04:01:02, but does not react with Cw3 antibodies. This allele was identified by sequence analysis, which indicated that a single G-to-A substitution at position 406 in exon 3 created a null allele under a new mechanism: the mutation changes the position of the intron 2-exon 3 splice site to be further into exon 3, leading to a frameshift and a premature stop codon. Sequence analysis of cDNA confirmed the existence of the causative alternative acceptor splice site and the resultant deletion of 64 nucleotides in exon 3. Analysis of 220 blood or bone marrow donors in Japan with C*03:23N demonstrated that Japanese HLA-C*03:23N is on the haplotype A*26:01∼C*03:23N∼B*40:02∼DRB1*09:01.

Autoantibody Profiles in Collagen Disease Patients with Interstitial Lung Disease (ILD): Antibodies to Major Histocompatibility Complex Class I-Related Chain A (MICA) as Markers of ILD.

Interstitial lung disease (ILD) is frequently associated with collagen disease. It is then designated as collagen vascular disease-associated ILD (CVD-ILD), and influences patients' prognosis. The prognosis of acute-onset diffuse ILD (AoDILD) occurring in patients with collagen disease is quite poor. Here, we report our investigation of auto-antibody (Ab) profiles to determine whether they may be useful in diagnosing CVD-ILD or AoDILD in collagen disease. Auto-Ab profiles were analyzed using the Lambda Array Beads Multi-Analyte System, granulocyte immunofluorescence test, Proto-Array Human Protein Microarray, AlphaScreen assay, and glutathione S-transferase capture enzyme-linked immunosorbent assay in 34 patients with rheumatoid arthritis (RA) with or without CVD-ILD and in 15 patients with collagen disease with AoDILD. The average anti-major histocompatibility complex class I-related chain A (MICA) Ab levels were higher in RA patients with CVD-ILD than in those without (P = 0.0013). The ratio of the average anti-MICA Ab level to the average anti-human leukocyte antigen class I Ab level (ie, MICA/Class I) was significantly higher in RA patients with CVD-ILD compared with those without (P = 4.47 × 10(-5)). To the best of our knowledge, this is the first report of auto-Ab profiles in CVD-ILD. The MICA/Class I ratio could be a better marker for diagnosing CVD-ILD than KL-6 (Krebs von den lungen-6).

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.

Relationship of donor HLA antibody strength to the development of transfusion-related acute lung injury.

BACKGROUND: Antibodies against the human leukocyte antigen (HLA) in donors' blood are implicated in the development of transfusion-related acute lung injury (TRALI). Screening of female donors for HLA antibodies has been introduced to prevent TRALI; however, the relationship of HLA antibody strength in the transfused components to the development of TRALI has not been evaluated in detail. STUDY DESIGN AND METHODS: Donors involved in 1038 cases of nonhemolytic transfusion reactions (NHTRs) including 283 cases of TRALI were screened for HLA antibodies by the fluorescence beads method. HLA antibody specificity and strength were analyzed in detail. The usefulness of enzyme-linked immunosorbent assay (ELISA) for screening HLA antibodies was also evaluated. RESULT: Among 21 cases of TRALI, four cases of possible TRALI, and five cases of other NHTRs, the sum of mean fluorescence intensity (MFI) of donors' HLA antibodies to patients' cognate antigen(s) was determined in 18, four, and three cases, respectively. The sum of MFI in TRALI cases was significantly higher than that in other NHTR cases (p<0.05). When HLA antibody-positive samples were reevaluated by ELISA, the ELISA optical density ratio was significantly higher in donors' samples associated with TRALI than in those associated with other NHTRs (p<0.01) CONCLUSIONS: A correlation between the HLA antibody strength and development of TRALI was indicated. The antibody strength measured by ELISA could be used as the basis for the screening of HLA antibodies in place of the fluorescence beads method. This study provided clues to the establishment of a cutoff value for HLA antibody screening in an evidence-based manner for the prevention of TRALI.

Survey of severe respiratory syncytial virus infection in Kyoto Prefecture from 2003 to 2007.

BACKGROUND: Respiratory syncytial virus (RSV) infection in infants can develop into a severe condition. METHODS: A survey of patients with severe RSV infection in hospitals in Kyoto Prefecture was performed from 2003 to 2007. Patients requiring intubation and those with cardiopulmonary arrest on arrival (CPAOA) were considered to have severe RSV infection. RESULTS: Twenty-five patients with severe infection were identified and detailed data were available for 21 patients, of whom 18 required intubation and three had CPAOA. The male/female ratio was 12/9 and age ranged from 8 days to 19 years (average, 5.2 months; median: 2 months). At admission white blood cell count, lactate dehydrogenase (P < 0.05), and blood glucose (P < 0.01) were higher and Na was lower (P < 0.01) in the 18 patients with severe infection (excluding the CPAOA cases) compared to 18 sex- and age-matched patients with mild RSV infection. The incidence of bacterial infection was also higher in severe cases (P < 0.05). The outcome was death in four patients (19.0%, including two sudden deaths), aftereffects in two (9.5%), hospital discharge with improvement in 14 (66.7%), and an unclear outcome in one patient. Excluding the two sudden death cases, 14 of 19 patients (73.7%) were extubated within 2 weeks. The period of intubation was longer in older patients (P < 0.05). CONCLUSION: Because severe RSV infection led to sudden death in two cases, detection of RSV is important at admission for an infant with CPAOA. Fourteen patients (66.7%), however, had good outcomes and most patients were extubated within 2 weeks.

Human leukocyte antigen matching estimations in a hypothetical bank of human embryonic stem cell lines in the Japanese population for use in cell transplantation therapy.

Human embryonic stem (hES) cell lines are of great potential in cell transplantation therapy. However, recipients of such allogeneic transplants probably need treatment with immunosuppressants. Recently, Taylor et al. [Lancet 2005;366:2019-2025] proposed banking of hES cell lines and estimated the required number of hES cell lines for beneficial human leukocyte antigen (HLA) matching in the U.K. population. Here, we carried out such an estimation in the Japanese population. We calculated the proportion of patients who can find at least one HLA-matched donor at three loci of HLA-A, -B, and -DR. With a bank of hES cell lines from 170 randomly selected donated embryos, 80% of patients were expected to find at least one hES cell line with a single mismatch at one HLA locus or a better match. Furthermore, 80% of patients are expected to find at least one donor with complete matching at the three HLA loci if parthenogenetic homozygous hES cell lines are established from 55 randomly selected donated oocytes. The relatively low ethnic diversity of the Japanese population may have resulted in a high success rate in beneficial matching. Moreover, parthenogenetic hES cell lines can greatly reduce the number required for a higher degree of HLA matching.

Unrelated cord blood transplantation for second hemopoietic stem cell transplantation.

BACKGROUND: The Kanagawa Cord Blood Bank (KCBB) reports the treatment of 12 patients who received umbilical cord blood transplantation (CBT) from unrelated donors as their second hemopoietic stem cell transplantation (HSCT). METHODS: Provided by the KCBB, between February 1997 and September 2000, 12 patients had unrelated CBT as a second HSCT. Six patients were male and six female; nine patients were in malignant, and three were in non-malignant conditions. The median age of the patients was 7.9 years (range, 2.2-28.0), and the median bodyweight was 22.5 kg (12.0-55.0). The HLA-A and -B serological and DR genotypical disparities between the patients and CBT donors were as follows: one patient was a 0-mismatch, six were 1-mismatches, and five were 2-mismatches. RESULTS: The median time between first and second HSCT was 14.0 months (1.0-47.0). The overall survival rate was 25.0%, three years after CBT (Kaplan-Meier estimate). Mortality after CBT as a second HSCT accounted for nine cases, six from infection and three from treatment-related mortality other than infection. CONCLUSION: Cord blood transplantation offers the advantage of rapid availability, absence of donor risk, and possibly less HLA restriction. In these contexts, unrelated CBT should be considered as a source of HSCT for a second transplant.