Manabu Yamaoka

Platelets expressing P-selectin and platelet-derived microparticles in stored platelet concentrates bind to PSGL-1 on filtrated leukocytes.

The levels of interleukin-6 and platelet-derived microparticles (PMPs) were measured in the blood of 137 pa tients with side effects from platelet concentrate (PC) transfu sion with leukocyte removal filtration, P-selectin-expressing platelet and PMPs in stored PC before and after the filtration, and filtered leukocytes positive for P-selectin glycoprotein li gand-1. The side effects, which were observed in 203 transfu sions for 84 patients with hematologic disease and 53 patients with nonhematologic disease with no significant difference be tween the two groups, included urticaria (75.9%), erythema (18.7%), and fever (17.2%), but no anaphylactic reactions. The levels of interleukin-6 and PMP correlated in both groups, and were significantly higher in the hematologic disease group than in the nonhematologic disease group. The level of PMP, but not interleukin-6, was significantly higher for patients testing posi tive for allergic reaction than for those testing negative. In the stored PC prior to filtration, the level of interleukin-6 was normal. The level of P-selectin-expressing platelets and PMPs was elevated before filtration, but was significantly lower after filtration. Taken together, the results suggest that PMP is in volved in the generation of transfusion reactions, and indicate that both platelets and PMP displaying P-selectin bind to P- selectin glycoprotein ligand-1 of leukocytes retained by the leukocyte filter.

Analysis of serum ErbB-2 protein and HLA-DRB1 in Japanese patients with lung cancer.

We investigated the relationship between ErbB-2 and HLA in order to clarify the clinical and genetic factors related to Japanese patients with lung cancer. Thirty-nine of the 73 lung cancer patients (53.4%) had elevated levels of ErbB-2. Only seven of 23 (30. 4%) patients with small cell carcinoma had elevated ErbB-2 levels. The prevalence of ErbB-2 positivity was highest (23 of 32; 71.8%) in patients with adenocarcinoma, while that in patients with squamous cell carcinoma was 50% (9 of 18). The frequencies of HLA A33, B44, B62, and B75 were lower in the lung cancer patients than in the control group. HLA-DR9 was higher in frequency in lung cancer patients than in the healthy controls (P<0.05), but HLA-DR6 was lower in frequency in lung cancer patients than in controls (P<0.01). DRB1*0901 was significantly higher in frequency in lung cancer patients than in controls (P<0.05). On the other hand, DRB1*0802, DRB1*1302 and the DRB1*14 group (*1401, *1403, *1405, *1406, and *1407) were completely absent in lung cancer patients. The frequencies of HLA B35, B52, B62, DRB1*0404, and DRB1*0406 were higher in the ErbB-2-positive lung cancer patients than in the ErbB-2-negative lung cancer patients. However, these types of HLA were not included in significant frequencies in our group of lung cancers. Our results suggest that some HLA-antigens/alleles participate in the pathogenesis of lung cancer in Japanese patients. In addition, the relationship between HLA-associated genetic factors and ErbB-2 seems to be weak. These findings suggest that ErbB-2 is correlated with prognostic factors for lung cancer independently of HLA-associated genetic factors.

A case of thrombotic thrombocytopenic purpura induced by acute pancreatitis

Thrombotic thrombocytopenic purpura (TTP) is a multisystemic microvascular disorder that may be caused by an imbalance between unusually large von Willebrand factor multimers and the cleaving protease ADAMTS13. In acquired TTP, especially in secondary TTP with various underlying diseases, the diagnosis is difficult because there are many cases that do not exhibit severe deficiency of ADAMTS13 or raised levels of ADAMST13 inhibitors. It is well known that collagen disease, malignancy, and hematopoietic stem cell transplantation can be underlying conditions that induce TTP. However, TTP induced by acute pancreatitis, as experienced by our patient, has rarely been reported. Our patient completely recovered with treatments using steroids and plasma exchange (PE) only. In cases where patients develop acute pancreatitis with no apparent causes for hemolytic anemia and thrombocytopenia, the possibility of TTP should be considered. Treatments for TTP including PE should be evaluated as soon as a diagnosis is made.

Antiglycoprotein IIb-IIIa autoantibody in a patient with immune thrombocytopenia after cord blood transplantation.

cells, containing 3.20 × 10 CD34 cells, per kilogram body weight. Hematological engraftment was confirmed 28 days after CBT. Complete chimerism was confirmed by the fluorescence in situ hybridization technique using sex chromosomes. On day 30, the patient’s platelet level was 12 × 10/μL, and the patient became thrombocytopenic and thereafter unresponsive to transfusion of platelets. Although she frequently received platelet transfusions, she died of cerebral bleeding (day 54). Using flow cytometry, the binding of immunoglobulin G (IgG) to the healthy platelets was examined in the patient’s sera. The extent of IgG binding increased in her sera after CBT compared with that before CBT (Figure. 2 A and B). Furthermore, Western blotting revealed that the patient’s sera following CBT had either anti-GPIIb or anti-GPIIIa autoantibodies (Figure. 2C). Immune-mediated thrombocytopenia following SCT can be seen in relation to infection and GVHD, and rarely as an autoimmune phenomenon due to immune dysregulation. Antiplatelet autoantibodies To the Editor: Persistent thrombocytopenia following stem cell transplantation (SCT) has been reported to have a poor survival prognosis. Most cases of SCT-related thrombocytopenia are considered to be associated with chronic graft-versus-host disease (GVHD). However, immune-mediated thrombocytopenia (ITP) following SCT has been suggested to be rare and occurs due to an underlying immune dysregulation mainly associated with GVHD. On the other hand, it has been reported that antiplatelet antibodies are detected in sera of the patients with ITP and that their major target antigen is platelet membrane glycoprotein (GP) IIb-IIIa. Furthermore, CD4 HLA-DR-restricted T cells of ITP proliferate when stimulated by GPIIb-IIIa tryptic peptides or membrane fragments but interestingly not by intact GPIIb-IIIa polypeptide. We herein present the first report of a patient with refractory anemia with excess blast (RAEB) in whom anti-GPIIb-IIIa antibodies were detected after cord blood transplantation (CBT). The patient was a 68-year-old Japanese woman with RAEB that was chemorefractory. There were no HLA-identical sibling donors, and an unrelated umbilical cord blood was prepared. It was confirmed that the patient and the cord blood mismatched at 3 HLA loci (patient: HLA-A 1101/2602, HLA-B 3501/5401, and HLA-DRB1 1501/0410; cord blood: HLA-A 1101/2601, HLA-B 5101/5401, and HLADRB1 1501/0405). The clinical course of CBT is shown in Figure 1. Fludarabine was administered at 35 mg/d from day −5 to day −2, whereas 4 Gy of total body irradiation was given in 2 fractions on day −1. For GVHD prophylaxis, the patient also received cyclosporin at 150 mg/d as a continuous infusion from day 0, 15 mg intravenous methotrexate on day 1 and 10 mg on days 3 and 6, and 45 mg/d prednisolone starting on day 11. In October 2005, she received 2.60 × 10 cryopreserved maternal cord blood stem Clinical and Applied Thrombosis/Hemostasis Volume 15 Number 1 February 2009 123-124

Genetic analysis of HLA, NA and HPA typing in type 2 diabetes and ASO

We examined the genetic status of human leucocyte antigens (HLA), human platelet alloantigens (HPA) and neutrophil‐specific antigens (NA) in patients with type 2 diabetes mellitus and diabetic arteriosclerosis obliterans (ASO). To our knowledge, the present study is the first report showing the relationship among three genetic factors in type 2 diabetes mellitus and ASO patients. HLA typing was performed by the polymerase chain reaction (PCR)–restriction fragment length polymorphism method. HPA‐typing and NA‐typing were by a PCR‐sequence‐specific primer method. The incidence of HLA‐DRB1*1501 was found to be significant in type 2 diabetes and non‐diabetic, particularly ASO‐positive patients, compared to control subjects. There were no differences in NA1/NA2 between the control and diabetic or non‐diabetic ASO groups. However, the frequency of NA2/NA2 in ASO‐positive diabetes and non‐diabetic ASO patients was significantly higher than controls. The a/b genotype of HPA‐5a/5b was significantly lower in type 2 diabetes and non‐diabetic ASO‐positive patients than in controls. These findings suggest that genetic studies of HLA, NA and HPA could be useful to understand the pathogenesis of type 2 diabetes and ASO.

Genetic Analysis of HLA- and HPA-Typing in Idiopathic (Autoimmune) Thrombocytopenic Purpura Patients Treated with Cepharanthin

We performed genetic analysis of human leukocyte antigen (HLA) and human platelet antigen (HPA) in 45 patients with cepharanthin-treated idiopathic thrombocytopenic purpura. HLA-typing was performed by the polymerase chain reaction-restriction fragment length polymorphism method, and HPA-typing by a polymerase chain reaction-sequence-specific primer method. There were 14 responders and 31 nonresponders. Responders included many patients who had already been treated with prednisolone. HLA-DRB1*0901 was significantly more common in responders than in nonresponders. In contrast, HLA-DRB1*0410 and DQB1*0401 were significantly more common in nonresponders. The a/b genotype of HPA-2a/2a (Ko(b)/Ko(b)) was significantly increased in responders. In contrast, HPA-2a/2b (Ko(b)/Ko(a)) and HPA-3a/3b (Bak(a)/Bak(b)) were significantly more common in nonresponders. These findings suggest that genetic studies of HLA and HPA can predict the response of idiopathic thrombocytopenic purpura to cepharanthin.

HLA and HPA Typing in Idiopathic Thrombocytopenic Purpura Patients Treated with Kami-kihi-to

We performed human leukocyte antigen (HLA) and human platelet antigen (HPA) in patients with Kami-kihi-to-responsive idiopathic thrombocytopenic purpura. The HLA-A2, A61 and Cw1 were significantly increased in responders compared with nonresponders, as were HLA DRB1 *0901, DRB1 *1502, and DPB1 *0501. In contrast, HLA DPB1 *0201 and DPB1 *0901 were significantly decreased in responders. The a/b genotype of HPA-2 and a/a genotype of HPA-3 were markedly increased in nonresponders, and anti-GPIb antibody was also increased. These results suggest that HLA, HPA, and anti-GP antibody studies may predict the response of idiopathic thrombocytopenic purpura to Kami-kihi-to.

Flow cytometric analysis of Tk-polyagglutination.

We used flow cytometry to investigate the Tk-polyagglutination that was observed in a patient with metastatic lung cancer (59-year-old man). We analyzed the fluorescent patterns of three types erythrocytes obtained from a patient, healthy type-0 subject, and neuraminidase-treatment using four kinds of FITC-conjugated lectins (Griffonia simplicifolia, GS-II; Dolichos biflorus, DBA; Glycine max, SBA; Arachis hypogaea, PNA). Furthermore, the reactivity of normal serum to the patients erythrocytes was analyzed by FITC-conjugated anti-human IgG and IgM antibodies. The patients erythrocytes bound to FITC-GS-II and -PNA, but neuraminidase-treated erythrocytes reacted to FITC-SBA and -PNA. These results were in accordance with those obtained using the aggregation method. However, the reactivity of normal serum to the patients erythrocytes according to flow cytometric analysis was different from that determined by the aggregation method. In particular, anti-human IgM antibody showed the well-reactive tendency. Our results suggest that flow cytometry was useful for the heterogeneous analysis of Tk-polyagglutination in a patient with malignant tumors.