A report on a modified protocol for flow cytometry-based assessment of blood group erythrocyte antigens potentially suitable for analysis of weak ABO subgroups.

BACKGROUND: Flow cytometry (FC) has proven its utility in scrutinizing AB antigen expression in red blood cells (RBCs), cooperating with serological tests for accurate blood group typing. However, technical difficulties may impair the characterization of weak ABO subtypes when background noises appear at non-negligible levels. STUDY DESIGN AND METHODS: We sought to establish an FC method that could prevent antibody-induced hemagglutination and an increase in cellular autofluorescence, two major issues inherent to RBC-FC analysis of AB expression. We optimized fixatives, multicolor-staining protocols, and sequential gating strategies. Blood samples from weak ABO subtype cases, Bm and Ael , were analyzed with the established protocol. RESULTS: The optimized mixture of glutaraldehyde and formaldehyde successfully generated fixed RBCs resistant to agglutination while maintaining low autofluorescence. These features allowed co-staining of leukocyte- and erythrocyte-markers, which enabled sequential gating strategies facilitating the precise AB antigen analysis in purely single RBCs with minimum background noises. By the established FC analysis, we could detect in the Bm sample a small RBC population exhibiting weak B antigen expression. The assay also proved it feasible to identify a small population (0.04%) of RBCs weakly expressing the A antigen in the Ael sample confirmed as harboring a rare c.816dupG ABO variant allele. CONCLUSION: The RBC-FC analysis described here allows the detection of AB antigens weakly expressed in RBCs while achieving minimum background noise levels in negative control samples. Overall, the modified protocol provides a quick and reliable assay valuable in transfusion medicine and is potentially applicable to the characterization of rare weak ABO variants.

Reduction in adverse transfusion reactions with increased use of washed platelet concentrates in Japan-A retrospective multicenter study.

Plasma removal by washing platelet concentrates (PCs) is effective in preventing adverse reactions to PC transfusions. The Japanese Red Cross Society (JRCS) started releasing washed PCs (WPCs) as a commercially approved blood product in September 2016. This retrospective multicenter study investigated the change in the number of transfused WPCs and the impact on the incidence of adverse reactions to PCs before and after the release. The numbers and types of transfused PCs and the adverse reactions to the PCs for a year before the start of the WPC release and for a year after the release were reported by 27 medical institutes in Japan. Transfusion information for approximately 8% of the amount of PCs supplied in Japan was analyzed during the study period. After the start of WPC release by the JRCS, the number of transfused WPCs doubled. The rate of adverse reactions to PCs decreased significantly (p = 0.0223), from 4.30% before the release to 4.05% after the release. The rates of adverse reactions to unwashed and WPCs were 4.13% and 0.84%, respectively. Allergic adverse reactions were significantly decreased after the release (3.60% before versus 3.37% after). No severe allergic reactions to WPCs were reported. The release of WPCs by the JRCS significantly reduced transfusion-related adverse reactions to PCs in Japan.

Clinical features and predictors of patients with critical limb ischemia who responded to autologous mononuclear cell transplantation for therapeutic angiogenesis.

The clinical features of patients with critical limb ischemia (CLI) who responded to angiogenesis using autologous peripheral blood mononuclear cell transplantation (PB-MNC) have not yet been fully characterized, and there are no useful predictors to judge the curative effect in the early period after PB-MNC. This study sought to clarify the clinical features and predictors in patients with CLI who were successfully treated using PB-MNC. 30 consecutive patients [arteriosclerosis obliterans: 24 patients, thromboangiitis obliterans: 6 patients] who were diagnosed with major amputation despite maximal medical therapy were enrolled in this study. The study endpoint was major amputation within 3 months after PB-MNC. The collected data were evaluated for correlation between patients with and without major amputation within 3 months after PB-MNC. Six patients underwent major amputation and 1 patient underwent minor amputation. In the patients with major amputation, transcutaneous oxygen tension before PB-MNC and transplanted CD34-positive cells were lower than those of patients without major amputation. In the patients with amputation, interleukin-6 (IL-6) continued to increase after the first PB-MNC, and basic fibroblast growth factor (bFGF) decreased within 3 days after the first PB-MNC. PB-MNC was useful for the patients who were managed for inflammation and who had revascularization of the upper-popliteal arteries and two of the infra-popliteal arteries by endovascular and/or surgical revascularization. Variation in IL-6 and bFGF in the early period after PB-MNC could be useful predictors for the requirement of amputation within 3 months after PB-MNC.

[Annual changes in antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli isolated from acute diarrheic patients].

Two hundred thirty-one Campylobacter were isolated from acute diarrheic patients between January 2001 and December 2005. We evaluated annual changes in identified species of Campylobacter and their susceptibilities against antibiotics. Campylobacter jejuni (219 strains; 94.8%) and Campylobacter coli (12 strains; 5.2%) were identified to the species. Susceptibilities to four antimicrobial agents, minocycline (MINO), levofloxacin (LVFX), erythromycin (EM) and clindamycin (CLDM) were examined. The resistant rates of four antimicrobial agents in C. coli were significantly higher than that in C. jejuni. The susceptibility of C. jejuni to LVFX was variable, and MICs gave a bimodal distribution. The resistant rate against EM was estimated to be 9.2% in C. jejuni, 66.7% in C. coli. Moreover, young people ranging from 19 to 24 years old were predominant (47.7%) among the Campylobacter enteritis patients.

[Evaluation of antigen diagnostic kit in group A streptococcus mass infection].

We report a Food-borne group A streptococcus epidemic at Kitasato University campus on July 30 and 31, 2005, believed caused by lunch. A current mass group A streptococcus infection differing from the food-borne epidemic above occurred at Kitasato University East Hospital, also believed caused by lunch. Group A streptococcus was detected using a prompt diagnostic kit and bacterial culture from 116 clinical specimens taken from 116 patients with group A streptococcus pharyngitis at Kitasato University East Hospital on August 5, 2005. To investigate the utility of immunochromatographic detection of group A streptococcus antigen, 116 clinical specimens obtained from pharyngeal membranes by swab were examined using a prompt diagnostic kit for group A streptococcus (ImmunoCard STAT! STREP A TEST) and conventional bacterial culture. Group A streptococcus positivity differed between the two methods. Fourteen patients were found to be positive by the prompt diagnostic kit and 23 by bacterial culture. Four patients showing 1.0 x 10(6) cfu/mL estimated by the culture were difficult to diagnose with the prompt diagnostic kit,even though the detection sensitivity of this kit was 1.0 x 10(6) cfu/mL or more. Conventional bacterial culture should therefore be used in addition to the prompt diagnostic kit to detect group A streptococcus, especially in pharyngeal samples obtained from patients with pharyngitis.

Expression of matrix metalloproteinase-2 and prostate-specific antigen in localized and metastatic prostate cancer.

BACKGROUND: The matrix metalloproteinases (MMP)-2 and MMP-9 are known to degrade the extra-cellular matrix (ECM) and to be involved in the invasion and metastasis of cancer cells. In order to establish the clinical significance of the determination of serum MMP-2 levels for prostate cancer (PCa), we determined serum levels of MMP-2 and PSA in patients with PCa at different stages. PATIENTS AND METHODS: In this study, serum samples from 39 patients with prostate hypertrophy and 51 PCa patients diagnosed at Kitasato University Hospital, Japan, were examined. MMP-2 levels were determined by measuring pro-MMP-2 using an enzyme immunoassay. PSA was also determined by enzyme immunoassay. Immunohistochemical analysis of prostate cancer specimens was performed according to the labeled streptavidin-biotinylated antibody (LSAB) method. RESULTS: Serum levels of MMP-2 and PSA increased with PCa disease progression. Serum levels of both markers in patients with invasive and metastatic PCa were significantly higher than in those with localized PCa. No significant association was found between serum levels of MMP-2 and PSA in patients with PCa. Extremely high levels of MMP-2 greater than 950 ng/ml or increased levels of PSA over 300 ng/ml were found only in PCa patients with bone metastases. CONCLUSION: Our findings indicate that MMP-2 may be involved in the invasion and metastasis of PCa and that serum MMP-2 levels may be able to become an auxiliary indicator of serum PSA to detect the progression of PCa.

Changes in serum IgG oligosaccharide chains with prostate cancer progression.

BACKGROUND: Changes of serum IgG oligosaccharide chain structure have been found in B cell lineage tumors and autoimmune diseases. Currently, the cancer-associated carbohydrate epitopes CA72-4 and CA15-3 are used as serum tumor markers. In the present study, we analyzed the structure of serum IgG oligosaccharide chains in prostate cancer (PCa) patients using the simple new method of fluorophore-associated carbohydrate electrophoresis (FACE). We also evaluated the relationship between changes of serum IgG oligosaccharide chain structure and serum concentration of prostate-specific antigen (PSA). MATERIALS AND METHODS: The structure of serum IgG oligosaccharide chains from 12 PCa patients (6 localized cancer, 6 metastatic cancer) and 10 healthy controls was evaluated by FACE. PSA levels in serum were determined by enzyme immunoassay. RESULTS: Fr 1 (monogalactosyl oligosaccharide) and Fr 2 (digalactosyl oligosaccharide) decreased significantly (p<0.05), while Fr 4 (agalactosyl IgG oligosaccharide) increased with PCa tumorprogression. The Fr 4/Fr 1+2 ratio in metastatic PCa patients was significantly higher than in healthy controls (p<0.05), and there was a significant correlation (r=0.84, p<0.05) between serum PSA levels and the Fr 4/Fr 1+2 ratio in all patients with PCa. CONCLUSION: The changes of serum IgG oligosaccharide chain structure with PCa progression are based on the abnormality of glycosylation in PCa metastasis. Therefore, the analysis of serum IgG oligosaccharide chain structure by FACE may be an auxiliary indicator of PSA for monitoring PCa progression.

Aortic valve replacement in a woman with osteogenesis imperfecta.

The mortality rate in cardiac surgery patients with heritable generalized connective tissue disorders, such as Marfan s syndrome and osteogenesis imperfecta, is high due to tissue friability. We describe a successful open heart surgery for repair of aortic regurgitation in a woman with osteogenesis imperfecta (OI). Although tissue friability caused no problems during surgery in this case, it should be kept in mind when operating on patients with OI.

[Current status of gene test market].

The technological innovation of the gene analysis makes the adaptation range of the gene test in clinical diagnosis expand. Then, gene test has popularized increasingly around the infection disease for clinical inspection. Also in the field of clinical inspection, the increase of the importance of clinical application and the inspection item new year by year have appeared with the functional analysis of a gene. Moreover, the new test method and automation analysis equipment tend to be developed by progress of gene-analysis technology, and it is going to be introduced. The spread of gene test and development of a gene test market have an important possibility of activating the present clinical inspection field.

Clinical usefulness of multiplex PCR lateral flow in MRSA detection: a novel, rapid genetic testing method.

Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I-V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3 h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.

Constitutive activity of nuclear transcription factor kappaB is observed in follicular lymphoma.

Constitutive activity of nuclear transcription factor kappaB (NF-kappaB) is observed in many pathological types of lymphoma that are associated with a poor clinical course. This suggests that NF-kappaB and pathways involving NF-kappaB are possible targets for successfully treating lymphoma. We examined 28 lymph nodes from 28 patients in whom follicular lymphoma was diagnosed from 1996 to 2006 at our institution, which were formalin-fixed and paraffin-embedded. The specimens were stained with an antibody that could recognize activated NF-kappaB and p65 to determine whether they were positive or negative for NF-kappaB activation. The clinical courses of the 28 patients were then correlated with the results of the NF-kappaB staining. The 10 men and 18 women had a mean age of 57.3 years (range, 25-87 years). By follicular lymphoma grade, 10 patients had grade 1, 16 had grade 2, and 2 had grade 3a. Ten patients died due to lymphoma. NF-kappaB was positive in 6 of the 28 cases. Analysis of the positive and negative staining groups while taking into account the clinical course, sex, age, grade of follicular lymphoma, prognostic index, CD10, CD23, Bcl-2, karyotype t(14;18), and survival showed that no significant differences. Six of the 28 lymph nodes (21.4%) exhibited consistent NF-kappaB activity. Three of the eleven cases that transformed to aggressive lymphoma were positive for activated NF-kappaB. Further research to clarify the significance of constitutive NF-kappaB activity in follicular lymphoma is therefore warranted.

CD3- and CD4-positive plasmablastic lymphoma: a literature review of Japanese plasmablastic lymphoma cases.

Plasmablastic lymphoma (PBL) is a very rare and recently-described subtype of diffuse large B-cell lymphoma. A maxillary tumor in an 84-year-old HIV-negative Japanese-man was referred. The biopsied specimen showed a diffuse proliferation of mature plasma cells, expressing CD3 (+), CD4 (+), CD20 (-), CD138 (+) and EBER (+) by immunohistochemistry. He was diagnosed as a plasmablastic lymphoma; radiation therapy (RT) was started, but the response to the RT was only a partial response. To our knowledge, this is the first report of a patient with PBL expressing CD3 and CD4.

Analysis of the oligosaccharide chain of human serum immunoglobulin g in patients with localized or metastatic cancer.

A quantitative imbalance between matrix metalloproteinases produced by cancer cells and tissue inhibitors of metalloproteinases produced by fibroblasts and other types of cells has been demonstrated to be a causative factor in invasion and metastasis of cancer cells. On the other hand, it is reported that sugar chains of adhesion molecules such as integrins and CD44 also influence the metastasis of cancer cells. Here, alterations of serum IgG oligosaccharide chain structure were investigated during tumor progression using the new method of fluorophore-assisted carbohydrate electrophoresis (FACE). The structure of serum IgG oligosaccharide chains from 22 cancer patients (11 localized cancer, 11 metastatic cancer) and 10 healthy controls was evaluated by FACE. It was clearly demonstrated that serum IgG oligosaccharide chains without galactose (agalactosyl IgG oligosaccharide) significantly increased with tumor progression of lung and gastric cancers. It is concluded that a marked increase of agalactosyl IgG oligosaccharide in these cancer patients is associated with carcinogenesis and metastasis. Therefore, the analysis of serum IgG oligosaccharide chain structure by FACE may be useful for evaluating diagnosis and prognosis in patients with these carcinomas.