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The detection of HLA antibodies is important in clinical practice, such as platelet transfusion refractoriness and transfusion-related lung injury. However, difficulties are associated with the preparation of panel cells for conventional HLA detection systems using intact cells, such as the immunocomplex capture fluorescence analysis (ICFA). Based on an ICFA analysis, HEK293 cells stably transfected with the HLA-A locus were used instead of peripheral blood mononuclear cells (PBMC). The reactivity, sensitivity, and stability of transfectants were examined. All 20 antisera to HLA-A identified by LABScreen® Single Antigen class I (LS-SA1) were reactive to our modified-ICFA (m-ICFA) and showed the same specificities as those in LS-SA1, indicating the cell surface expression and correct antigenicity of the HLA-A locus in transfectants. The expression of HLA class I antigens was similar between transfectants frozen for 6 years and those prior to freezing. In the reaction of the anti-A24 or anti-A33 antibody vs each transfectant, the index of m-ICFA was higher than that of WAKFlow® ICFA. Our m-ICFA also showed that false negative reactions sometimes observed in capture assays may be avoided. By using HLA-A transfectants as ICFA targets, we herein developed m-ICFA. Our m-ICFA may avoid false negative reactions of capture assay like enzyme-linked immunosorbent assay and can also be carried out in almost any laboratory without cell culture facilities.

Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p

Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p

Background Volume-reduced washed platelets (VR-wPLTs), which are prepared by concentrating platelets (PLTs) into a smaller volume of additive solution (AS), may prevent not only circulatory overload, but also adverse reactions caused by plasma components. Although VR-wPLTs may be quickly degraded due to high PLT concentrations, few studies have examined the effects of storage on VR-wPLTs. We examined here the in vitro properties of VR-wPLTs prepared with M-sol AS during their storage for 7 days. Study Design and Methods Platelet concentrates (PCs) were divided into two equal aliquots (control group and test group). After the centrifugation of both aliquots and removal of as much supernatant as possible, the pellet of the control group was resuspended in 160 mL of M-sol while that of the test group was resuspended in 80 or 40 mL of M-sol. The wPLTs of both groups were stored in polyolefin bags with agitation at 20 to 24°C for 7 days. Results The pH values of both groups were maintained at higher than 7.0 during the 7-day storage. Differences in %disk, CD62P, annexin V, percent hypotonic shock response, and aggregation values between the test group and control group were small for at least 2 days after washing. Conclusions The in vitro properties of VR-wPLTs were not markedly degraded for at least 2 days. Therefore, the storage properties of PLTs may be maintained in VR-wPLTs prepared at blood centers until they are administered to patients in hospitals.

Background To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma-derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA-B19V) in 2008. Study Design and Methods Donor samples that were positive by CLEIA-B19V screening were tested for B19V DNA. The sensitivity of CLEIA-B19V was tested using samples of all three genotypes and B19V DNA–positive donations. B19V DNA–positive donations and pooled plasma were quantitatively assayed for B19V DNA. B19V DNA–positive donations were phylogenetically analyzed by polymerase chain reaction direct sequencing. Results The sensitivity of CLEIA-B19V was inferred to be approximately 6.3 log IU/mL with the genotype samples and 6.4 log IU/mL with B19V DNA–positive donor samples. Of 417 CLEIA-B19V–positive samples from 1,035,560 donations in Hokkaido, Japan, 101 were positive for B19V DNA. The 198 strains of B19V DNA–positive donations in Hokkaido over the past 15 years clustered exclusively with Genotype 1. After introduction of CLEIA-B19V, the viral load for B19V DNA in all 772 pooled plasma for fractionation from donors in nationwide Japan did not exceed 4 log IU/mL. Conclusion CLEIA-B19V can detect all three genotypes of B19V (viral load >6.3 log IU/mL) and limit the viral load (

Hemoglobin vesicles (HbVs), artificial oxygen carriers encapsulating concentrated Hb solution on phospholipid vesicles (liposomes), are promising candidates for clinically useful transfusion. Although HbV infusion transiently suppressed the proliferative response of rat splenic T-cells to concanavalin A or keyhole limpet hemocyanin (KLH), a T-cell-dependent antigen, in ex vivo culture conditions, HbV infusion did not affect the primary IgG antibody response. We extended our assessment of the effects of HbV infusion on the systemic immune response using primary and secondary responses to KLH in rats. We observed that the generation of primary anti-KLH IgM antibody in HbV-infused rats was not suppressed but was instead higher than those in saline-infused rats. Furthermore, HbV infusion did not suppress the increase of IgG subclass of KLH antibody in secondary response. The T cell response to KLH of bulk spleen cells, as derived from 2-3 months after secondary KLH immunization, was unaffected by infusion of HbV, suggesting that HbV loading has no suppressive effect on homeostatic survival of memory T-cells against KLH. These results indicate that HbV is highly biocompatible in systemic immune responses in rats.

Putative animal reservoirs and environmental samples were studied to investigate potential routes of transmission for indigenous hepatitis E virus (HEV) infection in Hokkaido, Japan. A total of 468 liver samples and 954 environmental samples were collected from 2003 to 2011 for this study. Four swine livers (1 %) were positive for HEV RNA; two strains belonged to genotype 3 and the other two strains were genotype 4. Genotype 3 HEV was detected in a sewage sample and a seawater sample. HEV strains derived from swine liver, seawater and raw sewage samples shared 93-100 % sequence similarity with human HEV strains.

Background: Activated platelets form heterogeneous aggregates of platelets and monocytes, which are involved in a variety of inflammatory disorders. Some anti-human leukocyte antigen (HLA) Class I antibodies have been shown to activate platelets. Materials and Methods: Human leukocyte antigen-A2-positive or HLA-A2-negative platelets were incubated with HLA-A2-negative peripheral blood mononuclear cells (PBMNCs) in the presence of anti-HLA-A2 serum at 37°C. The binding of platelets to monocytes was analysed by flow cytometry. The levels of IL-1 β and IL-8 in the culture supernatant were determined by ELISA. Results: Anti-HLA-A2 serum increased the formation of aggregates between monocytes and HLA-A2-positive platelets, but not HLA-A2-negative platelets, in a dose-dependent manner. Antiserum also increased the number of platelets bound to monocytes in a time-dependent manner. The addition of anti-P-selectin glycoprotein ligand (PSGL-1) mAb almost completely inhibited the formation of platelet–monocyte aggregates as well as the number of platelets bound to monocytes. When HLA-A2-positive or HLA-A2-negative platelets were incubated with HLA-A2-negative PBMNCs in the presence of anti-HLA-A2, the level of IL-1β and IL-8 in the supernatant of coculture was significantly higher in HLA-A2-positive platelets than in HLA-A2-negative platelets. The addition of anti-PSGL-1 mAb partially but significantly inhibited the production of both IL-1β and IL-8. Conclusions: The activation of platelets with anti-HLA Class I alloantibody caused the formation of platelet–monocyte aggregates, followed by the production of IL-1β and IL-8, in a cognate antigen–antibody manner. The adhesive interaction of P-selectin and PSGL-1 at least partially contributed to these phenomena.

北海道函館赤十字血液センター（以下函館センター）の製剤部門は2006年に北海道赤十字血液センター（以下札幌センター）に集約され，管内供給は北海道ブロックの需給コントロールによって管理されることとなった．製剤部門の集約は，在庫量の少ない血小板製剤に影響が現れやすいと考えられたので，同製剤の緊急需要（当日受注）に対する供給実態について，受注から配送に至る経過に焦点をあて回顧的に調査した．当日受注で，在庫分に由来すると思われる配送所要時間が2時間未満の血小板製剤の割合は集約直後の2006年度で減少したものの，在庫見直し後の2009年度は2005年度並みに回復した．在庫分がなく札幌からの需給調整に由来すると思われる所要時間6時間以上の割合は2005年，2006年に比べ，2009年度では半減した．時間外発注で1時間以内に配送した割合も在庫見直し後の2009年度に有意に増加し88.5％に達した．製剤部門の存在は，血小板製剤の緊急需要に対し，一時的な在庫量の増加をもたらすものの安定供給の主要な要因とはならず，適正な在庫管理が最も重要な要因であることがわかった．血小板の緊急需要に対しては，通常の需要量を基礎にして在庫量を設定すること，需要量の変化に応じてそれを見なおすことにより対応が可能であった．供給規模が小さく，在庫管理の難しい地方血液センターでは，血小板製剤の広域の需給調整を活発にすることで経済効率を保ちつつ医療機関の需要に応えるべきと考える．

[背景]少子高齢化社会では赤血球製剤（RCC）の需要増加が見込まれる一方，献血者の減少が危惧される．現状の献血者の平均献血回数が年2回未満であることから，1回の採血赤血球量を増量し，現行の採血基準内（全血相当量1,200ml）で年2回採血が安全に実施できるかの検討は有意義と考える． [対象・方法]承諾を得た58kg以上の男性供血者18人から，赤血球成分採血（RCa）により3単位RCC（全血600ml相当の赤血球）を6カ月間隔で2回採血し，採血中・後の副作用および採血前と6カ月後まで血算，血清鉄，血清フェリチン（s-Ft），エリスロポエチンを検査した．RCaには，1回目（1-RCa）はヘモネティクス社CCS，2回目（2-RCa）は改良ボウルを組み込んだ同社のMultiを使用した． [成績]1-，2-RCaとも問題になる副作用はなく，Hb値は採血直後に11g/dl以上，3カ月後には採血前値に回復した．s-Ftは各採血前値に比し1-RCa 6カ月後61.8±20.2％，2-RCa 6カ月後77.0±29.5％の回復に留まったものの，経過中12ng/ml以下になった6例においてもHb値は回復した．2-RCa採血直後のRCCの遊離Hbは20.1±10.8mg/dlであった． [結論]3単位RCCを6カ月間隔で2回採血することは安全に実施できると考える．なお，s-Ftの動向は今後の検討課題と思われる．

濃厚血小板（PC）により引き起こされる輸血副作用の防止には洗浄血小板が有効である．血漿には菌の増殖を抑制する補体成分が含まれているため，洗浄により血漿濃度が減少した洗浄血小板中では，菌の増殖が促進される可能性がある．本研究では，M-solで調製した洗浄血小板中での菌の増殖動態を多血小板血漿（PRP）中でのそれと比較検討した． PRPに菌を播種し（Day0），20～24℃で24時間振とう保存した後，PRPを2等分（コントロール群とテスト群）した．テスト群の遠心上清を出来るだけ除去し，M-solを添加した（Day1）．両群はポリオレフィンバッグ中でDay7まで保存した．菌数測定は寒天培地を用いたプレート法により行った． PRP中での場合と比較すると，洗浄血小板中ではStreptococcus dysgalactiaeやEscherichia coliの増殖は促進され，Staphylococcus epidermidisとStaphylococcus aureusの増殖は抑制された．洗浄血小板中でのBacillus cereusの増殖はPRP中の場合とほとんど差がなかった．Propionibacterium acnesやSerratia marcescensの場合，PRPおよび洗浄血小板のいずれにおいても増殖しなかった． M-solで調製した洗浄血小板中で増殖が促進される菌株が存在するという点に注意が必要である．

【背景】輸血関連急性肺障害（transfusion-related acute lung injury：TRALI）の病因に，抗HLA Class II抗体による標的抗原陽性単球の活性化が関与していると考えられる．また，TRALIの特徴的な症状である肺水腫は血管透過性の亢進により誘導される． 【方法】TRALIにおける肺水腫の形成に抗HLA Class II抗体と単球が関与する可能性を調べるため，TRALIの原因製剤となった抗HLA Class II抗体陽性血漿（抗HLA-DR血漿）存在下で，ヒト末梢血単核細胞（peripheral blood mononuclear cell：PBMNC）とヒト肺微小血管内皮細胞（human lung microvascular endothelial cells：HMVEC）を共培養し，血管透過性亢進の有無を調べた．同様の実験を正常ヒト臍帯静脈内皮細胞（human umbilical vein endothelial cells：HUVEC）を用いて行った．血管透過性は，共培養に添加した蛍光標識デキストランの透過性を測定することにより評価した． 【結果】抗HLA-DR血漿存在下でPBMNCsとHMVECsまたはHUVECsを共培養することにより，血管内皮細胞の透過性が亢進した．この作用は抗体の特異性に依存していた．共培養にplatelet activating factor（PAF）のアンタゴニストであるCV-3988を添加することにより，血管透過性亢進作用はほぼ完全に抑制された．抗TNF-α中和抗体単独及び同抗体と抗IL-1β中和抗体の両方を共培養に添加することにより，HMVECsとHUVECの透過性亢進作用はそれぞれ部分的に抑制された． 【考察】抗HLA Class II抗体の存在下で標的抗原を発現する単球と血管内皮細胞を共培養することにより，血管透過性亢進が誘導された．この反応にPAF，TNF-αまたはIL-1βが関与していると考えられた．従って，抗HLA Class II抗体による単球の活性化は，TRALIにおける肺水腫の病態形成に関与することが示唆された．

【背景】日赤では，輸血用血液や血漿分画製剤原料へのヒトパルボウイルスB19（B19V）の混入を防止するため，化学発光酵素免疫測定法（chemiluminescence enzyme immunoassay：CLEIA）によるB19V抗原スクリーニング検査を実施している．近年，B19Vは3種の遺伝子型に分類されたため，米国食品医薬品局（FDA）は，血漿分画製剤製造工程中の検査として，すべての遺伝子型が検出可能な核酸増幅検査を導入するよう勧告した．しかし，本邦で流行しているB19Vの遺伝子型や，CLEIA法がB19Vの遺伝子型すべてを検出可能かは不明である．【対象と方法】既報を改変したB19Vユニバーサルreal-time PCR法（U-PCR法）およびCLEIA法で，3種類のB19V遺伝子型を含むWHOパネルを測定し，それぞれの検出感度を評価した．また，過去13年間の北海道内献血者から検出されたB19V陽性検体96例を用いて，U-PCR法による検出および遺伝子型を調査した．【成績】U-PCR法およびCLEIA法のいずれの方法でも，3種類のB19Vが検出可能であった．U-PCR法の検出感度は1型，2型および3型に対して，それぞれ13.6，9.4および14.6IU/ml以上であった．またCLEIA法の検出感度はいずれの遺伝子型についても約6.3 log10IU/ml以上と推測された．B19V陽性96例全例がU-PCR法で陽性となり，遺伝子型はすべて1型であった．【結論】CLEIA法は3種類のB19Vすべてを検出可能であった．また過去13年間に道内で検出されたB19Vは1型だけであった．

BACKGROUND: HLA Class II antibody–initiated activation of monocytes possessing the corresponding antigen is thought to participate in the pathogenesis of transfusion-related acute lung injury (TRALI). Pulmonary edema, a hallmark of TRALI, is caused by increasing vascular permeability. STUDY DESIGN AND METHODS: To investigate the contribution of HLA Class II antibody and monocytes to the development of pulmonary edema in TRALI, we studied whether the permeability of human lung microvascular endothelial cells (HMVECs) could be enhanced by coculturing HMVECs with peripheral blood mononuclear cells (PBMNCs) in the presence of HLA Class II antibody–containing plasma, which was implicated in TRALI (anti-HLA-DR plasma). In addition, similar experiments were performed with human umbilical vein endothelial cells (HUVECs). The endothelial permeability to fluoresceinated dextran, which was added from the start of coculture, was measured. RESULTS: The coculture of HMVECs or HUVECs with PBMNCs in the presence of anti-HLA-DR plasma resulted in the increase of endothelial permeability in the corresponding antigen-antibody–dependent manner. CV-3988, a platelet-activating factor (PAF) receptor antagonist, almost completely suppressed the increase in endothelial permeability. Neutralizing antibodies to tumor necrosis factor (TNF)-α alone and simultaneous addition of the antibodies to TNF-α and interleukin (IL)-1β to the coculture partially suppressed the permeability increase of HMVECs and HUVECs, respectively. CONCLUSIONS: HLA Class II antibody and monocytes in the corresponding antigen-antibody combination caused the enhancement of endothelial permeability. PAF, TNF-α, and/or IL-1β might be involved in the endothelial permeability increase. HLA Class II antibody–initiated monocyte activation could lead to the development of pulmonary edema in TRALI.

PTR is a serious problem in patients being treated for hematologic disorders. Two patients with acute leukemia developed PTR after allogeneic BMT from one HLA-antigen-mismatched mother attributable to HLA antibodies, which could not be detected in their serum before BMT. HLA antibodies, whose specificity resembled that of each patient, were detected in each donor's serum. Each donor had probably been immunized during pregnancy by their partner's HLA antigens expressed by the fetus, consequently, transplanted donor-derived cells provoked HLA antibodies in each recipient early after BMT, and those HLA antibodies induced PTR. If the mothers are selected as donors for their children, they should be tested for the presence of HLA antibodies.

Background and Objectives In previous studies, we reported the transmission of hepatitis E virus (HEV) by transfusion, and the frequent detection of HEV markers in Japanese blood donors with elevated ALT levels. For the current study, we carried out a nationwide survey of the prevalence of IgG anti-HEV in qualified blood donors throughout Japan. Materials and Methods The 12 600 samples from qualified blood donors were collected from seven blood centres (1800 per centre) representing nearly all regions of Japan. Samples were from age- and sex-matched blood donors who tested negative for all the current blood screening tests. The samples were screened using the in-house IgG anti-HEV ELISA. Sequentially, the positive samples were tested by the commercial IgG anti-HEV ELISA. Results Of 12 600 samples, 431 (3·4%) were regarded as positive for IgG anti-HEV. The prevalence of IgG anti-HEV was higher in eastern Japan (5·6%) than in western Japan (1·8%) (P

Background and Objectives The activation of neutrophils by human leukocyte antigen (HLA) Class I alloantibody is thought to be involved in transfusion-related acute lung injury. Neutrophils contain various biological substances in four groups of granules, including secretory vesicles, azurophilic granules, specific granules and gelatinase granules. To characterize the activation of neutrophils by HLA Class I alloantibody, we investigated whether HLA Class I alloantibody could cause the degranulation of these groups of granules either coordinately or selectively. Materials and Methods Sera containing HLA-A24 alloantibody were incubated with neutrophils in a washed whole blood system. CD11b expression (secretory vesicles) on neutrophils was analysed by flow cytometry, and the secretion of markers of each granule was determined by ELISA. Results The treatment of cross-matching-positive neutrophils with sera containing HLA-A24 alloantibody caused the significant expression of CD11b, and the significant secretion of neutrophil elastase and myeloperoxidase, azurophilic granule markers and heparin-binding protein (HBP), which is localized in secretory vesicles and azurophilic granules when compared with cross-matching-negative neutrophils. In contrast, no significant differences were observed in the secretion of lactoferrin, a marker of specific granules, and matrix methalloproteinase-9, a marker of gelatinase granules between cross-matching-positive and cross-matching-negative cells upon stimulation with sera. CD11b expression and secretion of HBP by serum was partially inhibited by p38 mitogen-activated protein (MAP)-kinase inhibitors. Conclusion Neutrophils activated with sera containing HLA Class I alloantibody caused the preferential degranulation of azurophilic granules and secretory vesicles. This process was at least in part mediated by p38 MAP kinase-involved signal transduction.

洗浄・置換血小板（W/R-PC）は血小板製剤による輸血副作用を防止する目的で調製されている．我々は血小板製剤の洗浄および置換には臨床使用が認められた輸液および電解質溶液を混和させることで製造される洗浄置換液（M-sol）を用いている．M-solの製造はクリーンルームにて用時行っているが，本研究では以下の二点を中心に製造方法の改良を行った．1）除菌フィルター付バッグを使用することで，クリーンルーム外での製造を可能とした．2）調製したM-solはアルミ蒸着袋に真空保存することで，pHを一定に保つことが可能となり最短でも1年間の保存が可能であった．さらに我々はW/R-PC 1バッグ分のM-solを製造する方法（単品式）と一度に多量のM-solを製造する方法（多量式）を考案し，両法によるM-solに差がないことを確認した．真空包装したM-solは30℃で1年間保存してもpHに変化はなかった．さらに，製造直後のM-solと長期保存したM-solにより調製された置換PC（R-PC）には血小板機能に差はみられなかった．また，長距離輸送したM-solを用いて調製した洗浄PC（W-PC）についても，血小板への影響はみられなかった．改良された方法ではM-solの製造時間に制限されずにW/R-PCの調製が可能となるであろう．

Dombrock血液型のDoaとDob抗原は，共優性の対立遺伝子DOA，DOBによって決定される．何れの抗原も免疫原性は高くなく，抗体が検出されることはまれである．抗Doaと抗Dobは，通常，反応性が弱く，他の抗体と共存しているため，同定が非常に難しい抗体である．したがって，抗原陰性血のタイピングに利用可能な抗血清の入手は困難であり，通常は交差適合試験によって適合血が選択される．しかし，その適合血の信頼性は高くなく，患者に溶血性輸血副作用をもたらすことがある． 今回我々は，抗Doa保有患者の輸血において，血清学的タイピングに替わる方法としてPCR-SSP法を用いたDNAタイピングにより適合血の選択を行った．患者にはDOA陰性（DOB/DOB）の2バッグが輸血されたが，溶血所見は認められなかった．なお，北海道内の献血者（n=235）におけるDombrock遺伝子の頻度は，DOAが0.109，DOBが0.891であった． Dombrock血液型のDNAタイピングは，適合血のスクリーニングにおいて有用な方法であると考えられた．

Dentinogenesis imperfecta (DGI) type II is one of the most common dominantly inherited dentin defects, in which both the primary and permanent teeth are affected. Here, we report a Japanese family with autosomal-dominant DGI type II, including both molecular genetic defects and pathogenesis with histological analysis. Mutation analysis revealed a mutation (c.53T>A, p.V18D, g.1192T>A) involving the second nucleotide of the first codon within exon 3 of the dentin sialophosphoprotein (DSPP) gene. This mutation has previously been reported in a Korean family. Thus far, 24 allelic DSPP mutations have been reported, and this is the seventh mutation involving the DSPP V18 residue. Among those, only one other was shown to be caused by a de novo mutation, and that mutation also affected the V18 amino acid residue. The DSPP V18 residue is highly conserved among other mammalian species. These findings thus suggest that the V18 amino acid might be a sensitive mutational hot spot, playing a critical role in the pathogenesis of DGI.

BACKGROUND: Type I allergic reactions such as urticaria-like manifestations constitute a large percentage of transfusion-related adverse events. Along with donor factors, patient factors might be involved in these reactions. Sera from some patients with chronic idiopathic urticaria show histamine-releasing activity (HRA). Sera from patients who develop Type I allergic reaction might possess HRA. STUDY DESIGN AND METHODS: Pretransfusion serum samples were collected. Mast cells were cultured from peripheral blood CD34+ cells and mixed with the serum samples. Cells with elevated intracytoplasmic Ca2+ concentrations were monitored using flow cytometry to evaluate Ca2+ influx–inducing activity (CaIA) in serum. The amount of histamine released into the supernatant was measured using an enzyme immunoassay kit to evaluate HRA. In some assays, cells were incubated with pertussis toxin (Ptx). RESULTS: CaIA values were higher (p

Vascular endothelial cells regulate the passage of fluids, solutes, and cells from the vascular space to the tis- sues. Disruption of vascular integrity is involved in the pathogenesis of inflammatory diseases including transfusion- related acute lung injury (TRALI), a most severe nonhemolytic transfusion reaction with symptoms such as dyspnea and/or hypotension and fever. Pulmonary edema, due to increased vascular permeability for macromolecules and plasma, is a hallmark of TRALI. The mortality rate of TRALI ranges from 5 to 10%. While donor antibodies (Abs) against human leukocyte antigen (HLA) class I and granulocytes are regarded as causative factors, various clinical studies have demon- strated the roles of anti-HLA class II-Ab on the etiology of TRALI, although the detailed mechanisms have not been clari- fied. Over several years we have investigated to clarify the underlying mechanism by which anti-HLA class II Abs cause an increase in endothelial permeability. In this review, we show that anti-HLA class II Ab generates proinflammatory cy- tokines and chemokines from HLA class II positive mononuclear cells of peripheral blood in an Fc R-dependent manner. As a result, the produced interleukin-1 and tumor necrosis factor- lead to increased endothelial permeability via the nu- clear factor- B pathway but not apoptosis of endothelial cells. These findings provide a better understanding of the roles of anti-HLA class II Ab in the etiology of TRALI.

BACKGROUND: Leukodepletion reduces but does not eliminate adverse reactions to platelet concentrate (PC). As an alternative strategy, plasma reduction or washing of platelets should be considered. However, the efficacy of this strategy is still unclear. STUDY DESIGN AND METHODS: A total of 12 patients who experienced adverse reactions at a 29 to 100 percent reaction rate for plasma-PC were enrolled. The reactions were allergic reactions and nonhemolytic transfusion reactions, such as chills. Plasma-removed PC (W/R-PC), which was suspended in a recently developed additive solution (M-sol) containing less than 20 mL plasma, was prepared. W/R-PCs in M-sol were then transfused into patients after an overnight storage period; the occurrence of adverse reactions was monitored and 1- and 24-hour corrected count increment (CCI) values were evaluated. RESULTS: Although plasma-PC caused reaction in 12 patients, W/R-PC prevented reactions in 11 of 12 patients, with 1 patient having one minor allergic reaction of 15 transfusions. There was a significant difference in the incidence of reaction (p

Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development as clinically useful blood substitutes. Although HbVs are shown to distribute transiently into the bone marrow in animal models, the influence of HbVs on human hematopoietic stem/progenitor cells has not yet been studied. Therefore, we investigated the influence of HbVs at a concentration of up to 3 vol/vol % on the clonogenic activity (in semisolid culture) and proliferative activity (in liquid culture) of human hematopoietic progenitor cells derived from umbilical cord blood (CB) in vitro. Continuous exposure of CB mononuclear cells to HbVs tended to decrease the number and size of mature-committed colonies and most notably reduced the number of colonies of high-proliferative potential colony-forming cells (HPP-CFC). In contrast, exposure to HbVs for 20 h or 3 days, which is more relevant to the clinical setting, had no effect on the number of mature-committed colonies and only modestly decreased the number of HPP-CFC. Continuous exposure (10 days) to HbVs significantly suppressed the cellular proliferation and differentiation of both the erythroid and myeloid lineages in liquid culture. Again, short exposure (20 h or 3 days) did not affect these parameters. Thus, our results show that HbVs, under conditions relevant to the clinical setting, have no adverse effect on human CB hematopoietic progenitor activity in vitro.

Background: To overcome the shortage of blood and blood products, it is necessary to increase the total volume of blood collected from donors. This requires modifying the current standard of 200-ml whole-blood collection for young people (16-17 year-old), which is specified by the Departmental Regulation No.22 (Kouseishou-rei dai-22 gou). The objectives of this study were to evaluate the safety of 400-ml whole-blood collection in 17-year-old males. Methods: A total of 322 male volunteers aged 17 years and 363 aged 18 or 19 years were recruited for blood collection through advertisements at schools and blood center donation sites. Blood collected from the 17-year-old males was not used for transfusion, whereas that collected from the 18-19 year-olds was used for transfusion as normal. To evaluate the safety of 400-ml whole-blood collection in the 17-year-old males, Fisher's exact test was used to compare vasovagal reaction (VVR) occurrence rates between the two groups by using a two-by-two contingency table. Significance was defined as P 0.05). The recovery rate of cell counts and plasma components to the original level was similar between the two groups. We conclude that 400-ml whole-blood collection can be performed safely in 17-year-old males in Japan.

血小板輸血による副作用を予防する目的で血漿を除去した洗浄·置換血小板（Washed and/or replaced platelet concentrates，W/R-PC）が調製されている．調製法としては2つの方法が主に使われているが両者を比較した報告はない．そこで，成分採血由来の血小板（Plasma-PC）を洗浄置換·保存液M-solで洗浄し，M-solに再浮遊する方法（洗浄置換法WR-method）とplasma-PC中の血漿を遠心後M-solに置換する方法（置換法R-method）について，各W/R-PCの血小板機能や輸血効果を比較検討した． 各方法で調製されたW/R-PCの血小板回収率や残存血漿蛋白，in vitroにおける性状（pH，凝集能，%HSR，P-セレクチン陽性率，血小板形態）を調べ，アナフィラキシーショックなどの重篤な副作用がみられた患者を対象に，WR-methodによるPC（WR-PC）を75バッグ（患者: 6名），R-methodによるPC（R-PC）を31バッグ（患者: 4名）に投与して，補正血小板増加数（CCI）及び副作用の予防効果を比較した． WR-PCとR-PCの血小板回収率はそれぞれ90.5±1.4%と89.5±1.8%で有意差はなく，血漿蛋白除去率（残存蛋白量）は，WR-PCでは96.9±0.7%（428±95mg）で，R-PCの95.4±0.9%（627±130mg）より有意に高かった（各n=7）．48時間の保存期間中（PC採血後5日目），in vitroの性状はpH以外の項目に差はみられなかった．Plasma-PCは保存と共にpHが低下し，24時間後には7.05±0.04であったのに対し，W/R-PCは何れも調製直後に一時的なpHの低下が認められたが，WR-PCでは7.37±0.03，R-PCは7.40±0.02に上昇した．輸血24時間後のCCI（×104/ul）は，WR-PCが1.53±0.82（n=51），R-PCが1.59±0.78（n=18）と良好であり，何れのW/R-PCについても，輸血後の副作用の発生はなく，副作用予防効果が示された．また，何れも有害事象は認められなかった． WR-PCとR-PCは，血小板機能や輸血効果について，同等である事が確認されたが，R-methodは調製工程が少なく，より簡便であるため，PCの血漿除去法として推奨される．

赤血球保存液を添加した解凍赤血球濃厚液（FTRC）について，in vitroにおける長期保存試験を実施した．冷凍赤血球（FRC）およびFTRCの製造には血球洗浄装置ACP215（ヘモネティクス）を用いた．はじめに，FTRCの製造法を検討したところ，Meryman改良法はValeri法よりも洗浄効果が高いことが示された．次に，保存液として米国で用いられているAS-3液，または国内での使用が認められているMAP液をFTRCに添加·保存し，その性状を調べた．AS-3を添加したFTRCの製造後7日目と14日目の溶血率はそれぞれ1.0±0.2と1.2±0.2%であった．MAP液を添加したFTRCの製造後7日目と14日目の溶血率はそれぞれ0.8±0.2%と1.4±0.3%であった．これらの値を現行のFTRCの12時間目の溶血率（2.9±1.4%）と比較すると，はるかに低値であることを確認した． ACP215を用いてMAP液を加えたFTRCを製造した場合，米国食品医薬品局の溶血許容値（溶血率1%未満）を参考にすると，その有効期間を最長7日間へ延長する事が可能であると考えられた．

Background The generation of inflammatory mediators from monocytes activated by HLA Class II antibodies is thought to play important roles in the etiology of nonhemolytic transfusion reactions. Increased permeability of endothelial cells contributes to the pathogenesis of rash, urticaria, angioedema, and pulmonary edema, which are symptoms of transfusion reactions. Study design and methods We investigated whether inflammatory mediators released from monocytes upon stimulation by HLA Class II antibodies could increase endothelial permeability. Human endothelial cell monolayers were incubated with cell-free supernatants of peripheral blood mononuclear cells (PBMNCs) stimulated with HLA Class II antibody-containing plasma (anti-HLA-DR plasma), which has been implicated in severe nonhemolytic transfusion reactions. The permeability of endothelial cells to dextran was measured. Results The supernatants of PBMNCs stimulated with the anti-HLA-DR plasma in corresponding antigen-antibody combinations were able to increase endothelial permeability. At least 3 hours of exposure of PBMNCs to anti-HLA-DR plasma was required to produce a supernatant that could induce a significant increase in permeability. Simultaneous addition of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) neutralizing antibodies to the activated PBMNC supernatant significantly reduced the increase in permeability. Treatment of the endothelial cells with an inhibitor of nuclear factor kappaB (NF-kappaB), but not inhibitors of apoptosis, significantly prevented the increase in permeability. Conclusion Both TNF-alpha and IL-1 beta, generated from PBMNCs by anti-HLA-DR plasma in a corresponding antigen-antibody-dependent manner, led to an increase in endothelial permeability. The activation of monocytes by the HLA-DR antibodies and the resultant inflammatory mediators could contribute to the pathogenesis of rash, urticaria, angioedema, and pulmonary edema after transfusion.

BACKGROUND: Five cases of transfusion transmission of hepatitis E virus (HEV) have been reported so far. The infection routes of the causative donors remain unclear, however. Also, the progress of virus markers in the entire course of HEV infection has not been well documented. STUDY DESIGN AND METHODS: Nucleic acid testing was performed by real-time reverse transcription-polymerase chain reaction targeting the open reading frame 2 region of HEV. Full-length nucleotide sequences of HEV RNA were detected by direct sequencing. RESULTS: Lookback study of a HEV-positive donor revealed that the platelets (PLTs) donated from him 2 weeks previously contained HEV RNA and were transfused to a patient. Thirteen relatives including the donor were ascertained to enjoy grilled pork meats together in a barbecue restaurant 23 days before the donation. Thereafter, his father died of fulminant hepatitis E and the other 6 members showed serum markers of HEV infection. In the recipient, HEV was detected in serum on Day 22 and reached the peak of 7.2 log copies per mL on Day 44 followed by the steep increase of alanine aminotransferase. Immunoglobulin G anti-HEV emerged on Day 67; subsequently, hepatitis was resolved. HEV RNA sequences from the donor and recipient were an identical, Japan-indigenous strain of genotype 4. HEV RNA was detectable up to Day 97 in serum, Day 85 in feces, and Day 71 in saliva. CONCLUSION: A transfusion-transmitted hepatitis E case by blood from a donor infected via the zoonotic food-borne route and the progress of HEV markers in the entire course are demonstrated. Further studies are needed to clarify the epidemiology and the transfusion-related risks for HEV even in industrialized countries.

血小板製剤による輸血副作用を発症する患者に対し，洗浄置換血小板（W/R-PC）を使用する場合がある．我が国では数種類の洗浄置換液が使用されているが，血小板の保存性能などを詳細に検討した報告はほとんどなく，W/R-PCの有効期限なども統一されていない．最近われわれは血小板保存性能の優れた洗浄置換液（M-sol）が市販輸液製剤のみで調製できることを明らかにした．本研究では現在我が国で広く使用されている洗浄置換液3種とM-solについて血小板保存性能の比較検討を行った．さらにM-solの液状での保存方法についても検討を行った． M-sol，ブドウ糖加酢酸リンゲルを主とする液，生理食塩水+ACD-A，冷凍血液洗浄用液3号+ACD-AによりW/R-PCを96時間まで保存した．測定項目であるpH, Pセレクチン陽性率，%HSR，%Disk, MPV，凝集能，グルコース，乳酸の数値から判断すると，洗浄置換後6時間以内ならばいずれの洗浄置換液中でも血小板機能は良好であったが，24時間を超えて保存する場合はM-solの場合のみ通常のPCと同等もしくはそれ以上の機能が維持されていた．またM-solの液状保存に関しては，アルミ蒸着した袋を用いて4℃で静置することにより，少なくとも3カ月間安定に保存できることが明らかになった．

To prevent the risk of infection by infusions of plasma products contaminated with infectious agents in medical treatments, a nanofiltration technique is available to eliminate the agents in the process of plasma manufacture. In this study, the capacity for microparticle removal of the virus filter membrane (Planova®), which consists of hollow fibers (BMMs), for nanofiltration, was estimated by electron microscopy using 20 nm colloidal gold and human parvovirus B19 (HPVB19) solutions, and plasma specimens from an HPVB19-patient. The BMMs used were of 35 nm (BMM35), 20 nm (BMM20) and 15 nm (BMM15) mean pore size. To determine the site at which particles were retained, the BMM wall cut transversely was divided into 10 sections from inner to outer surface of the membrane on the electron micrographs. When gold particle solutions (3.6 × 1011 particles/0.5 ml) were filtered with 5 mm-length single modules, particles were retained in the section 10, 7, and 8 in the wall of BMM35, BMM20, and BMM15, respectively. When purified HPVB19 suspensions (109–1013 TCID50-infectivity/ml in 0.5 ml) were filtered using the modules, virus particles were observed in the section 10, 5, and 7 in BMM35, BMM20, and BMM15, respectively. After the plasma filtration (107 TCID50), virus particles were also observed in the section 8, 4, and 3. Virus particles were identified by immunoelectron microscopy using an anti-HPVB19 monoclonal antibody. In the BMM20 used for HPVB19 filtration, many immunogold particles were counted on or around the virus observed in the section 5, but the number was drastically reduced and no virus was observed in the outer sections. No infectious HPVB19 was detected in the filtrates from BMM20 and BMM15. These results demonstrate that BMM20 and BMM15 can completely remove microparticles larger than 20 nm from the solution and that the smallest human viruses such as HPVB19 can be removed by using the nanofiltration technique.

BACKGROUND: Japanese Red Cross (JRC) blood centers implemented anti-hepatitis B core antigen (HBc) screening in 1989 and 50-minipool (MP)-nucleic acid testing (NAT) in 2000. A systematic lookback study has been conducted to determine the hepatitis B virus (HBV) transmission risk of donations drawn in the pre-hepatitis B surface antigen (HBsAg) and/or MP-NAT window phase and by donors with occult HBV infection. STUDY DESIGN AND METHODS: JRC blood centers have been storing aliquots of every blood donation since 1996. On the basis of the complete repository tube archives, all donations from repeat donors received from 1997 to 2004 were subjected to a lookback study. When repeat donors turned positive for HBV viral marker(s), repository tubes from their previous donations were tested for HBV with individual-donation (ID)-NAT. The frequency of ID-NAT-only–positive donations and the HBV transmission risk by the transfusion of those components were investigated. RESULTS: HBV ID-NAT was performed on 15,721 repository tubes, and 158 tubes (1.01%) were found positive for the presence of HBV DNA. Of these 158 ID-NAT-only–positive donations, 95 (60%) were derived from carriers with low anti-HBc titers. Of 63 patients transfused with ID-NAT-only–positive components, 12 (19%) proved to be infected with HBV. Only 1 of 33 components with low anti-HBc titers could be identified as infectious, whereas 11 of 22 anti-HBc–negative components proved to be infectious. None of the 16 identified hepatitis B surface antibody–positive components showed serologic evidence of infection. CONCLUSION: The clinically observed HBV infection risk caused by blood components from occult HBV carriers with low anti-HBc titers who slip through the JRC screening system is more than 10-fold lower than the transmission risk by donations in the pre-HBsAg and/or MP-NAT window phase.

BACKGROUND: To reduce adverse reactions due to platelet (PLT) transfusion, medical solutions on the market, such as saline and ACD-A, are used to replace the plasma of PLT concentrates in Japan; however, they are not strongly preservative. Here, an attempt was made to develop a novel additive solution (M-sol) having the ability to preserve PLTs stably, with only approved solutions for clinical use. STUDY DESIGN AND METHODS: M-sol is a mixture of solutions for medical use, which consists of 77 mmol per L NaCl, 3 mmol per L KCl, 1 mmol per L CaCl2, 21 mmol per L Na acetate, 15 mmol per L glucose, 9.4 mmol per L Na3 citrate, 4.8 mmol per L citric acid, 44 mmol per L NaHCO3, and 1.6 mmol per L MgSO4. The in vitro variables of PLTs stored in M-sol, Seto-sol, PASIIIM, or 100 percent plasma were compared during 14 days of storage. RESULTS: The in vitro parameters (pH, P-selectin, %hypotonic shock response, %disk, mean PLT volume, aggregability) of PLTs were better maintained in M-sol containing 3 percent plasma than in 100 percent plasma, PASIIIM with 31 percent plasma, and Seto-sol with 3 percent plasma during 14 days of storage. CONCLUSION: The 2-week storage of PLTs in M-sol is feasible in terms of the in vitro PLT function. Our results here show that the additive solution, with a high ability to preserve PLTs, can be prepared by mixing solutions approved for clinical use that are not specifically for PLT storage.

The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA. However, the amount of sc-DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6-4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 8-Oxo-7,8-dihydro-2'-deoxyguanosine did not form in the M13 genome after UV-C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV-C irradiation under these conditions. These results indicated that UV-C irradiation induced not only CPD and (6-4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV-C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.

BACKGROUND: Antibodies to neutrophil antigens have been implicated in neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. Most often, neutrophil-specific antibodies are directed toward human neutrophil antigen (HNA)-1 (Fcγ receptor 3b) and HNA-2a (CD177) in these disorders. STUDY DESIGN AND METHODS: To detect the alloantibodies in the serum samples, a panel of cell lines was established in which the HNA-1a, HNA-1b (polymorphisms of HNA-1), or HNA-2a gene was transduced with a retrovirus vector to confer stable transgene expression in K562 cells that exhibited low background reactivity to human serum samples obtained from healthy donors in flow cytometric analysis. RESULTS: It was shown that several well-characterized human serum samples containing antibodies against HNA-1a, -1b, and -2a were unambiguously identified by the established panel cell lines and observed a lower background reactivity and longer shelf life of the K562 panel cell lines compared with isolated neutrophils, which have been used for the cell panel to identify antibodies against HNA in human serum samples. CONCLUSION: These results indicate that the K562 panel cell lines provide a good panel for detecting HNA-reactive neutrophil antibodies in human serum samples.

BACKGROUND: There has been controversy over whether HLA alloimmunization is a risk factor for platelet (PLT) transfusion refractoriness (PTR) in hematopoietic peripheral blood progenitor cell transplantation (HPBPCT). STUDY DESIGN AND METHODS: Reported here is a boy with relapsed leukemia who developed fatal PTR after a peripheral blood progenitor cell transplantation (PBPCT) as a second HPBPCT from his mother. To elucidate the cause of PTR, a single-antigen assay (FlowPRA, One Lambda), a magnetic particles mixed passive hemagglutination test, and anti-human immunoglobulin-lymphocyte cytotoxicity test were performed on serum samples of the patient and his mother. RESULTS: Although HLA Class I antibodies were absent in his serum sample before HPBPCT, the serum sample after the first bone marrow transplantation (BMT) reacted weakly with beads coated with multiple HLA Class I molecules. After PBPCT, the positive reaction markedly increased. Although HLA-B44 antibody emerged transiently after BMT, the apparent generation of antibodies against HLA-A2 and -A24 as well as HLA-B44 occurred after PBPCT. The continuous appearance of HLA Class I antibodies coincided with the duration of marked PTR after PBPCT. The patient, however, had no antibodies against PLT-specific glycoproteins. Unidentified HLA Class I–reactive antibodies were detected in maternal serum sample. CONCLUSION: Although the patient appeared to be immunized to allogeneic HLA Class I molecules after BMT, profound HLA alloimmunization might have occurred after PBPCT in this case. It is possible that the administration of large numbers of immunocompetent cells sensitive to alloantigens at PBPCT causes the aberrant and persistent production of the HLA Class I antibodies.

大量化学療法施行後に, B型肝炎ウイルス (HBV) が再活性化したと考えられる輸血患者を経験した. 多発性骨髄腫と診断された50歳代の女性に対し自己末梢血幹細胞移植を含む大量化学療法を施行した際, 赤血球濃厚液を20単位, 血小板濃厚液を305単位使用した. 治療1年後 (2005年9月) に輸血後感染症検査を行ったところ, HBV-DNAが陽性であった. 治療前 (2002年8月) と輸血継続中 (2004年2月) のHBs抗原が陰性であったため, 輸血によるHBV感染を疑い, 2003年9月から2005年4月までの患者保管検体 (合計12本) で検査を行った. 輸血前 (2003年9月) の検体では, HBsAg (-), HBsAb (+), HBV-DNA (-) であったがHBcAb (+) で, 潜在性HBV感染と推定された. 以降, 2004年9月までは同様の検査結果であったが, 2005年4月にはHBs抗原の陽性化とともに血清中にHBV-DNAが確認され, HBVの再活性化と診断. HBs抗体の力価は, 2003年9月から行われた大量化学療法に一致し徐々に低下, 2005年4月には陰性化した. HBV陽性の期間, ウイルス再活性化による肝炎は発症していない. 輸血血液保管検体の個別核酸増幅検査はすべて陰性で, 輸血によるHBV感染は否定された. 本症例では輸血後感染症検査によってHBV感染が初めて認識され, 輸血前を含む保管検体の遡及調査で, HBV再活性化にいたる変化を追跡し得た. 輸血後にはじめてウイルス感染が確認された場合には, その原因をつきとめるために輸血前検体の保存が必須である.

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4(+)/CD8(+) T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions.

Background HLA Class II antibodies are thought to be involved in severe transfusion reactions including transfusion-related acute lung injury (TRALI). The activation of monocytes by HLA Class II antibody may play an important role in the etiology of TRALI. Case report An 81-year-old man with non-Hodgkin's lymphoma (Clinical Stage IIIA) received a plateletpheresis unit containing at least 4 x 10(11) platelets because of thrombocytopenia and a bleeding tendency. Approximately 30 minutes after the start of transfusion, he developed chills, tachycardia, dyspnea, lumber, and abdominal pain and then a fever (40.3 degrees C). His SaO(2) dropped to 70 percent. The transfusion was discontinued immediately. His symptoms disappeared after treatment with oxygen and the administration of corticosteroid and aminophyrine. A chest X-ray showed no sign of pulmonary edema. Results The donor serum sample had HLA-DR antibodies against multiple DR antigens including DR13, the recipient's HLA-DR type. The cross-match between the patient's lymphocytes and the donor serum was positive. The treatment of peripheral blood mononuclear cells from healthy subjects bearing DR13 antigen with the donor plasma caused the secretion of inflammatory cytokines (i.e., interleukin [IL]-1beta, IL-6, and tumor necrosis factor-alpha) and neutrophil-activating chemokines (i.e., IL-8 and CXCL1/GRO-alpha) in a cognate antigen-antibody relationship. In addition, the secretion of inflammatory cytokines appeared to require the involvement of CD32 and/or CD16. Conclusion HLA-DR antibodies, detected in this case, had biologic functions to induce production of not only inflammatory cytokines but also neutrophil-attractant chemokines in vitro, which could contribute to the etiology of severe nonhemolytic transfusion reactions.

ヒトパルボウイルスB19 (以下B19) に感染した人の献血によって, B19が輸血用血液や分画原料血漿に混入し, 更にその製剤を介して患者にB19が感染する可能性がある. その危険性を減少させるため, 日本赤十字社ではReceptor-mediated hemagglutination assay (RHA) 法による献血者B19スクリーニングを導入した. それにより分画プール血漿中に混入するB19量は大幅に減少した. しかしFDAなどは原料プール血漿に混入するB19DNA量を104geq/mL (IU/mL) 以下とするよう勧告しており, RHA法の検出限界である1010IU/mLでは, この要求を十分に満たすものではない. 今回我々は新しく感度のよいchemiluminescence enzyme immunoassay for screening B19 (CLEIA-B19抗原) を開発した. B19DNA陽性パネル (n=152) をCLEIA-B19抗原で測定した結果, RHA法より4Log高感度であることがわかった. また20プールNAT陰性の671検体をCLEIA-B19抗原で測定した結果, 1例のみ偽陽性となった. 以上のことよりCLEIA-B19抗原は感度特異性に優れ, 献血者B19スクリーニングに有用であると考えられた.