Characteristics of False-Positive Alarms in the BacT/Alert 3D System.

The BacT/Alert system has been used for detecting the presence of bacteria in various clinical settings as well as in blood services, but it is associated with a relatively high incidence of false-positive results. We analyzed the results of our quality control sterility testing of blood products by BacT/Alert 3D to understand the mechanism of false-positive results. Anaerobic and aerobic bottles were inoculated with 10 mL of samples and cultured in BacT/Alert 3D for 10 days. Positive-reaction cases were classified as true positive if any bacterium was identified or false positive if the identification test had a negative result. The detection algorithm and the bottle graph pattern of the positive reaction cases were investigated. Among the 43,374 samples, 25 true positives (0.06%) and 29 false positives (0.07%) were observed. Although the detection algorithm of all true positives and 25 of 29 false positives was accelerating production of CO2, a steep rise in the bottle graph was observed only in the true positives, and it was not observed in either of the false positives. Four of 29 false positives were dependent on high baseline scatter reflections. Furthermore, evaluating the bottle graph pattern of Streptococcus pneumoniae, a bacterium known to autolyze, we confirmed that no viable bacterium was detected even if a steep rise was observed. In conclusion, the bottle graph pattern of positive reactions allows the differentiation between true positives and false positives. In case of a steep rise without bacterium detection, the bacterium might have autolyzed. Moreover, positive reactions with high baseline scatter reflections, despite immediate loading of bottles after sampling, are potentially false positive. IMPORTANCE In clinical settings, false-positive results are treated as positive until bacterial identification. It may result in the discarding of blood products in blood centers or affect clinical decisions in hospitals or testing facilities. Moreover, the management of these samples is usually time- and labor-consuming. The results of our study may help clinicians and laboratory staff in making a more precise evaluation of positive reactions in BacT/Alert.

Universal Detection of Mia Antigen and Frequencies of Glycophorin Hybrids among Blood Donors in Taiwan by Human Monoclonal Antibodies against Mia (MNS7), Mur (MNS10), and MUT (MNS35) Antigens.

Glycophorin hybrids such as GP.Mur are common in Southeast Asians. In Taiwan, clinically significant alloantibodies to the GP.Mur phenotype are the most important issue in blood banks. A large-scale screening of glycophorin hybrids in the Taiwanese population is urgently needed to ensure transfusion safety. Four clones of human hybridomas that secrete anti-Mia, anti-MUT, and anti-Mur were established by fusing human B-lymphocytes and myeloma cells (JMS-3). The specificity of each monoclonal antibody (MoAb) was characterized. Three MoAbs were applied on an Automated Pretransfusion Blood Testing Analyzer (PK7300/PK7400) for donor screening. Genotyping was performed to determine the detailed subgrouping of glycophorin hybrids. Four MoAbs are IgM antibodies. Anti-Mia (377T) binds to 46DXHKRDTYA54, 48HKRDTYAAHT57 peptides, and anti-Mia (367T) binds to 43QTNDXHKRD51 peptides (X indicates T, M, or K). Anti-Mur is reactive with 49KRDTYPAHTA58 peptides. Anti-MUT is reactive with 47KHKRDTYA54. A total of 78,327 donors were screened using three MoAbs, and 3690 (4.71%) were GP.Mur, 20 (0.025%) were GP.Hut, and 18 (0.022%) were GP.Vw. When the Mia antigen was introduced as routine screening, the frequency of Mi(a+) among blood donors in Taiwan was 4.66% (67,348/1,444,541). Mia antigen was implemented as a routine blood testing, and the results were labeled on all red blood cell (RBC) units.

Perioperative Allogeneic Blood Transfusion Is Associated With Surgical Site Infection After Abdominoperineal Resection-a Space for the Implementation of Patient Blood Management Strategies.

Allogeneic blood transfusion (ABT) has been reported as a major risk factor for surgical site infection (SSI) in patients undergoing colorectal surgery. However, the association of ABT with SSI in patients undergoing abdominoperineal resection (APR) and total pelvic exenteration (TPE) still remains to be evaluated. Here, we aim to elucidate this association. The medical records of all patients undergoing APR and TPE at our institution in the period between January 2000 and December 2012 were reviewed. Patients without SSI (no SSI group) were compared with patients who developed SSI (SSI group), in terms of clinicopathologic features, including ABT. In addition, data for 262 patients who underwent transabdominal rectal resection at our institution in the same period were also enrolled, and their data on differential leukocyte counts were evaluated. Multivariate analysis showed that intraoperative transfusion was an independent predictive factor for SSI after APR and TPE (P = 0.004). In addition, the first-operative day lymphocyte count of patients undergoing APR, TPE, and transabdominal rectal resection was significantly higher in nontransfusion patients compared with transfusion ones (P = 0.026). ABT in the perioperative period of APR and TPE may have an important immunomodulatory effect, leading to an increased incidence of SSI. This fact should be carefully considered, and efforts to avoid allogeneic blood exposure while still achieving adequate patient blood management would be very important for patients undergoing APR and TPE as well.

Prediction of the preoperative chemoradiotherapy response for rectal cancer by peripheral blood lymphocyte subsets.

BACKGROUND: Although neoadjuvant chemoradiotherapy (CRT) has become a standard procedure to downstage locally advanced rectal cancer prior to surgery, markers to predict the response to CRT have not been fully identified. The aim of this study was to identify predictive factors of response to CRT, especially focusing on peripheral blood leukocyte subsets. METHODS: A total of 45 consecutive patients diagnosed with primary rectal cancer were prospectively enrolled and received CRT followed by curative resection. The numbers of each lymphocyte subset in peripheral blood pre- and post-CRT were analyzed using flow cytometry. According to the pathological response to CRT, patients were classified into high (Hi-R) and low (Lo-R) response groups. RESULTS: Hi-R cases had significantly higher numbers of pre-CRT lymphocytes (p = 0.018), T lymphocytes (p = 0.009) and helper T lymphocytes (Th lymphocytes, p = 0.015) compared to the Lo-R cases. With the receiver-operating characteristic curve for numbers of pre-CRT T lymphocytes, the area under the curve (AUC) was 0.733, and the optimal cutoff value was 1196/µl, with 76.5% sensitivity, 67.8% specificity, 59.1% positive and 82.6% negative predictive values. The numbers of pre-CRT Th lymphocytes and cytotoxic lymphocytes were both independent predictors of the high CRT response in the multivariate analysis. CONCLUSIONS: In addition to the direct cytotoxicity of CRT, recent studies have demonstrated the induction of an immunological host response, which also contributed to the tumor regression induced by CRT. Our result suggested the potential role of circulating T lymphocytes in predicting the response to CRT in colorectal cancer patients.

Phosphatidylserine-specific phospholipase A1 (PS-PLA1) expression in colorectal cancer correlates with tumor invasion and hematogenous metastasis.

BACKGROUND: The function of phosphatidylserine-specific phospholipase A1 (PS-PLA1), a phospholipase that acts specifically on phosphatidylserine and produces lysophosphatidylserine, a lysophospholipid mediator, has not been fully elucidated. We evaluated the role of PS-PLA1 in oncogenesis and metastasis of colorectal cancer (CRC). MATERIALS AND METHODS: Specimens from 85 patients with CRC were immunostained with a monoclonal antibody against PS-PLA1. The correlation between PS-PLA1 expression and the clinicopathological variables was analyzed. RESULTS: Tumor depth and hematogenous metastasis independently positively correlated with PS-PLA1 expression. High PS-PLA1 expression was associated with shorter disease-free survival, although it was not an independent predictive factor. CONCLUSION: PS-PLA1 expression in CRC is associated with tumor invasion and metastasis.

Characteristics and prognosis of colorectal cancer associated with rheumatic disease.

It is well known that host immunity plays an important role in the defense against colorectal cancer (CRC) progression. The effects of autoimmune diseases, such as rheumatic disease (RD) in which the immune system is deregulated, on this immunity have not been fully investigated. The medical records of 1299 consecutive patients diagnosed with primary colorectal cancer who underwent surgical resection were retrospectively reviewed. The clinicopathologic factors of 28 subjects with RD (RD group) were compared with those of 1271 patients without RD (non-RD group). Compared to the non-RD group, the RD group was typified by a predominance of females (P < 0.01), older age (P < 0.01), and a lower incidence of rectal cancer (P = 0.02). Although no difference was observed between the groups in terms of TNM classification, disease-free and overall survival were significantly poorer in the RD group in both univariate and multivariate analyses. Subjects who had RD for more than 10 years tended to have a higher frequency of lymph node metastasis (P = 0.06) and a significantly higher incidence of synchronous distant metastasis (P = 0.035) at the time of cancer diagnosis. RD was associated with a significantly poorer prognosis of colorectal cancer, suggesting that deregulation of the immune system by autoimmune diseases may adversely affect the host immune defense against colorectal cancer progression.

Immunohistochemical detection of high-mobility group box 1 correlates with resistance of preoperative chemoradiotherapy for lower rectal cancer: a retrospective study.

BACKGROUND: High-mobility group box 1 (HMGB1) is a nucleoprotein that is related to inflammation. It has been implicated in a variety of biologically important processes, including transcription, DNA repair, differentiation, development, and extracellular signaling. Recently, its important role in the process of tumor invasion, metastasis, and resistance to anti-cancer therapies has been demonstrated. In this study, we aimed to investigate the correlation of HMGB1 expression and resistance of rectal cancer patients to chemoradiotherapy (CRT) prior to curative operation. METHODS: We retrospectively reviewed the data of 75 lower rectal cancer patients without complete pathological response who had received preoperative CRT and had undergone curative resection at the University of Tokyo Hospital between May 2003 and June 2010. HMGB1 expression in surgically resected specimens was evaluated using immunohistochemical detection and specimens were classified into high or low HMGB1 expression groups. Clinicopathologic features, degree of tumor reduction, regression of tumor grade, and patient survival were compared between the groups using non-paired Student's t-tests and Kaplan-Meier analysis. RESULTS: A total of 52 (69.3%) patients had high HMGB1 expression, and 23 (30.7%) had low expression. HMGB1 expression was significantly correlated with histologic type (P=0.02), lymphatic invasion (P=0.02), and venous invasion (P=0.05). Compared to patients with low HMGB1 expression, those with high expression had a poorer response to CRT, in terms of tumor reduction ratio (42.2 versus 28.9%, respectively; P<0.01) and post-CRT histological tumor regression grade (56.5 versus 30.8% grade 2; respectively; P=0.03). However, no significant correlation was found between HMGB1 expression and recurrence-free and overall survival rates. CONCLUSIONS: HMGB1 expression may be one of the key factors regulating the response of rectal cancer to preoperative CRT in terms of tumor invasiveness and resistance to therapy.

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

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

Platelet-derived sphingosine 1-phosphate induces migration of Jurkat T cells.

BACKGROUND: The migration of T cell to atherosclerotic lesions is proposed to be involved in the pathogenesis of the atherosclerosis. Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from activated platelets, exerts a variety of responses such as cell migration and proliferation, and reportedly induces T cell migration. Accordingly, platelet-T cell interactions may exist based on T cell responses triggered by platelet-derived S1P. METHODS: S1P was measured using two-step lipid extraction followed by high-performance liquid chromatography (HPLC) separation while other phospholipids were determined by an enzymatic assay. The expression of S1P and lysophosphatidic acid receptors on Jurkat T cells was examined by RT-PCR and flow cytometry. Jurkat cell migration by S1P and the supernatant of activated platelets (SAP) was evaluated by a modified Boyden's chamber assay. RESULTS: S1P1 receptor was confirmed to be expressed on Jurkat T cell by RT-PCR and flow cytometry. S1P at 10-100 nM induced strong Jurkat cell migration, which was inhibited by the S1P1 (and S1P3) antagonist VPC23019 and the Gi inactivator pertussis toxin (PTX). We found that the supernatant (releasate) of human platelets activated by collagen stimulation, which contains S1P abundantly, induced Jurkat cell migration and that the migration was inhibited by VPC23019 and PTX. In addition, human serum, into which platelet contents (including S1P) are fully released, induced the Jurkat cell migration, which was also inhibited by VPC23019. CONCLUSIONS: Our findings suggest that platelet-derived S1P induces Jurkat T cell migration possibly via S1P1. S1P may be a key molecule involved in the responses triggered by platelet-T cell interactions, including atherosclerosis.

Proximal shift of colorectal cancer along with aging.

INTRODUCTION: Although several reports have documented the increased incidence of right-sided colorectal cancer (CRC) in the elderly, especially in women, the gender-specific, age-related changes in the characteristics of CRCs, especially related to the cancer localization, have not been fully investigated. This study evaluated the age-related changes in the clinicopathologic features of CRCs, according to the gender. MATERIALS AND METHODS: A total of 1059 consecutive patients with CRCs who were admitted to the authors' surgical department between February 2005 and June 2012 were retrospectively reviewed. The patients were divided into male (n = 632) and female (n = 427) groups and then according to the age group, and the correlation between the age group and the other clinicopathologic features was analyzed by univariate and multivariate analysis. RESULTS: The number of concomitant adenomas found was significantly increased along with increasing age in men, and the presence of concomitant adenoma was the only independent age-related factor of male CRC in the multivariate analysis (P = .0044). In contrast, in women, the location of the CRC progressively shifted to the right side (proximal colon) with increasing age, and the presence of right-sided CRC was the only independent factor of female CRC in the multivariate analysis (P < .0001). CONCLUSION: There was a significant gender-specific difference in the age-related changes in the characteristics of CRC. Increasing the number of concomitant adenomas and the shift of CRC localization to the proximal colon were the gender-specific characteristics of male and female CRC, respectively.

Novel swine model of transfusion-related acute lung injury.

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

Barium enema and CT volumetry for predicting pathologic response to preoperative chemoradiotherapy in rectal cancer patients.

BACKGROUND: Preoperative chemoradiotherapy has been widely used for the prevention of local recurrence of locally advanced rectal cancer, and the effect of chemoradiotherapy is known to be associated with overall survival. OBJECTIVE: We aimed to evaluate the association of the pathologic response grade with tumor recurrence rate after chemoradiotherapy, using radiographic analysis and the Response Evaluation Criteria in Solid Tumors as the parameters. DESIGN: This study was conducted at a single tertiary care institution in Japan. SETTING: This was a retrospective cohort study of patients undergoing preoperative chemoradiotherapy. PATIENTS: A total of 101 low rectal cancer patients receiving preoperative chemoradiotherapy from July 2004 to August 2012 were enrolled. MAIN OUTCOME MEASURES: The tumor reduction rate was measured with the use of traditional Response Evaluation Criteria in Solid Tumors, barium enema, and CT volumetry, and the correlation between the reduction rate and the pathologic response grade was examined. RESULTS: The tumor reduction rate assessed according to Response Evaluation Criteria in Solid Tumors showed no association with the pathologic response grade (p =0.61). In contrast, the radiographic response rate by both barium enema and CT volumetry strongly correlated with the pathologic response grade (p < 0.0001 and p = 0.001).In terms of local tumor recurrence, those diagnosed as high responders by the pathologic response grade, Response Evaluation Criteria in Solid Tumors, barium enema, and CT volumetry had a lower recurrence rate (p =0.03, p =0.03, p =0.0002, and p =0.001). The difference between high responders and low responders was especially prominent by barium enema and CT volumetry. LIMITATIONS: The study is limited by its retrospective nature. CONCLUSIONS: Double-contrast barium enema and CT volumetry were superior to Response Evaluation Criteria in Solid Tumors in evaluating the effect of chemoradiotherapy and predicting the likelihood of tumor recurrence.

Temsirolimus and chloroquine cooperatively exhibit a potent antitumor effect against colorectal cancer cells.

PURPOSE: Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored. METHODS: We evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo. RESULTS: In vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio. CONCLUSIONS: Based on these data, we propose TEM with or without chloroquine as a new treatment option for CRC.

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

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

Changes in the plasma levels of cytokines/chemokines for predicting the response to chemoradiation therapy in rectal cancer patients.

In the present study, we aimed to characterize the predictive value of cytokines/chemokines in rectal cancer (RC) patients receiving chemoradiation therapy (CRT). Blood samples were obtained pre- and post-CRT from 35 patients with advanced RC, who received neoadjuvant CRT followed by surgery, and the correlation between plasma levels of cytokines/chemokines and the response to CRT was analyzed. The pre-CRT levels of soluble CD40-ligand (sCD40L) and the post-CRT levels of chemokine ligand-5 (CCL-5) were significantly associated with the depth of tumor invasion and with venous invasion. In addition, a significant decrease in sCD40L and CCL-5, as well as in platelet counts, was associated with a favorable response to CRT. A significant correlation between pre-CRT platelet counts and sCD40L was observed in patients with a favorable response. By contrast, higher post-CRT interleukin (IL)-6 was associated with a poor response. Platelets, immune system and cancer cells, cross-linked through various cytokines/chemokines, appear to play an important role in the response to CRT, and by understanding their roles, new approaches for the improvement of the therapy might be proposed.

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

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