Anti-A and anti-B titers, age, gender, biochemical parameters, and body mass index in Japanese blood donors.

It has been reported that anti-A and anti-B (ABO antibody) titers decrease with age, but little is known about the association between ABO antibody titers and physiologic/biochemical parameters such as body mass index (BMI), gamma-glutamyl transpeptidase (GGT), and total cholesterol (T-Cho). We investigated the present situation of ABO antibody titers among healthy blood donors in Japan and the physiologic/biochemical factors that may be associated with changes in ABO antibody titers. Plasma from 7450 Japanese blood donors was tested for ABO antibody titers using ABO reverse typing reagents by an automated microplate system; donor samples were classified into low, middle, and high titers according to the agglutination results obtained with diluted plasma samples. Multivariate regression analysis was performed to analyze the association between ABO antibody titers and age, gender, biochemical parameters (alanine transaminase [ALT], GGT, globulin, T-Cho, and glycosylated albumin [GA]), and BMI according to the ABO blood groups. A significant correlation between ABO antibody titers and age/gender, except for gender in anti-A of blood group B donors, was observed. BMI showed significant but negative correlations with anti-A and anti-B (ß = -0.085 and -0.062, respectively; p < 0.01) in blood group O donors. In addition, significant but negative correlations between GGT and T-Cho with anti-B of blood group A donors (ß = -0.055 and -0.047, respectively; p < 0.05) were observed. Although differences existed among the ABO blood groups, ABO antibody titers seem to be associated with physiologic and biochemical parameters of healthy individuals.

Regulation of the lysophosphatidylserine and sphingosine 1-phosphate levels in autologous whole blood by the pre-storage leukocyte reduction.

BACKGROUND AND OBJECTIVES: The effect of leukoreduction and storage periods on the accumulation of bioactive lysophospholipids and substances in human autologous blood (AB units) has not been fully investigated. MATERIALS AND METHODS: The accumulation of bioactive lysophospholipids such as sphingosine 1-phosphate (S1P) and lysophosphatidylserine (LysoPS) in AB units during the storage was investigated. The time-dependent changes and the effect of the filtration in pre-storage leuckoreduction (LR) and unmodified samples derived from 46 AB units were analysed. Additionally, the changes of lysophospholipids and platelet releasate, namely ß-thromboglobulin (ß-TG), induced by exposure of whole blood (WB) or platelet-rich plasma (PRP) to the filter material were analysed. RESULTS: LysoPS, but not S1P levels, time-dependently and significantly increased in both unmodified and LR samples. LysoPS significantly decreased in LR compared with unmodified samples, whereas S1P increased in LR compared with unmodified samples. In addition, exposure of WB and/or PRP to the filter material in vitro resulted in increased levels of S1P, LysoPS and ß-TG. CONCLUSIONS: LR effectively reduced the accumulation of LysoPS in AB units. On the other hand, it increased concentrations of S1P due to platelet activation by exposure to the filter material. These suggest that increases of S1P levels in LR and LysoPS in the unmodified samples were mainly caused by the leukocytes and/or platelets and that LR was effective in inhibiting the accumulation of LysoPS.

Perinatal management of neonatal alloimmune thrombocytopenia associated with anti-group A antibody.

OBJECTIVE: To prevent neonatal alloimmune thrombocytopenia due to anti-group A antibody perinatal management was performed. BACKGROUND: We previously reported a case of severe intracranial haemorrhage associated with neonatal alloimmune thrombocytopenia due to anti-group A isoantibody. MATERIAL/METHODS: A 40-year-old Japanese woman, gravida 4 para 1, was pregnant with her second baby. The previous sibling developed severe thrombocytopenia and died 10 days after birth due to intracranial haemorrhage. He was diagnosed with neonatal alloimmune thrombocytopenia; the causative antibody was found to be the anti-group A antibody. Prednisone was started at 7 weeks' gestational age. Intravenous immunoglobulin 1 g kg(-1) week(-1) was started at 29 weeks' gestational age and continued to delivery. Serological studies and genotyping were performed. RESULTS: The second boy was delivered at 33 weeks' gestational age by caesarean section. He was discharged without intracranial haemorrhage or thrombocytopenia. The anti-group A antibody titre in the maternal serum was 2048-4096 (normal range: 4-64). The anti-group A antibody titre in the newborn's serum was 4. Cross-matching between the maternal serum and the paternal platelets was positive. CONCLUSION: Owing to the history of neonatal alloimmune thrombocytopenia causing intracranial haemorrhage and death of the previous sibling, strict follow-up of the subsequent pregnancy was conducted.

Surgical stress response after colorectal resection: a comparison of robotic, laparoscopic, and open surgery.

BACKGROUND: The perioperative immune status of colorectal robotic surgery (RS), laparoscopic surgery (LS), and open surgery (OS) patients has not been compared. Our aim was to evaluate perioperative stress and immune response after RS, LS and OS. METHODS: This prospective study included 46 colorectal surgery patients from the Department of Surgical Oncology of the University of Tokyo Hospital. Peripheral venous blood samples were obtained preoperatively and on postoperative days 1, 3, and 6. We evaluated expression of HLA-DR (marker of immune competence), C-reactive protein (CRP) levels, and lymphocyte subset counts (natural killers, cytotoxic T cells and helper T cells). RESULTS: Fifteen, 23, and 8 patients underwent RS, LS and OS, respectively. HLA-DR expression was the lowest on day 1 and gradually increased on days 3 and 6 in all the groups. There was no significant difference in postoperative HLA-DR expression between the RS and LS group. However, on day 3, HLA-DR expression in the RS group was significantly higher than in the OS group (p = 0.04). On day 1, CRP levels in the LS group were significantly lower than in the RS group (p = 0.038). There were no significant perioperative changes in the lymphocyte subset cell count between the three groups. CONCLUSIONS: Perioperative surgical stress, as evaluated by immunological parameters, was comparable between robotic and laparoscopic surgery and higher with open surgery. Robotic surgery may be an alternative to laparoscopic surgery, as a minimally invasive surgery option for colorectal cancer.

The frequencies of human neutrophil alloantigens among the Japanese population.

Human neutrophil antigens (HNAs) play an important role in a variety of clinical conditions including immune-mediated neutropenia, non-hemolytic transfusion reactions, and transfusion-related acute lung injury. The aim of this study was to investigate the frequency distribution of HNAs-1 to -5 among the Japanese population. We analyzed samples from 570 healthy Japanese by molecular and serologic techniques to estimate the gene frequencies of HNAs-1 to -5. DNA samples were obtained and typed for the HNA-1 (n = 523), -3 (n = 570), -4 (n = 570), and -5 (n = 508), by molecular techniques. The HNA-1 genotype was determined by using a commercial polymerase chain reaction-reverse sequence-specific oligonucleotide probes (PCR-rSSOP) kit. The HNA-3 to -5 genotypes were determined by the PCR-sequence specific primer (PCR-SSP), previously described, with a small modification. The HNA-2a phenotype was determined in 301 donors by granulocyte immunofluorescence test. In Japanese, the gene frequencies of HNA-1a, -1b, and -1c were 0.623, 0.377, and 0.000, respectively. The frequency of HNA-2a phenotype was 0.987, and the gene frequencies of HNA-3a and -3b were 0.654 and 0.346, respectively. HNA-4a and -4b were found at 1.000 and 0.000, respectively, and HNA-5a and -5b at 0.840 and 0.160, respectively. We describe, for the first time, the frequencies of all HNAs (HNA-1 to -5) among the Japanese population. This study will be helpful for the prediction of the risk of alloimmunization to HNA, especially to determine the risk of HNA alloantibody production by transfusion of HNA incompatible blood and feto-maternal incompatibility.

Recommendations of the ISBT Working Party on Granulocyte Immunobiology for leucocyte antibody screening in the investigation and prevention of antibody-mediated transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is currently one of the most common causes of transfusion-related major morbidity and death. Among the many TRALI mediators, leucocyte antibodies have been identified as important triggers of severe TRALI. STUDY DESIGN AND METHODS: These recommendations were compiled by experts of the ISBT Working Party on Granulocyte Immunobiology, based on the results obtained in eight international granulocyte immunology workshops, their personal experiences and on published study results. RESULTS: Leucocyte antibody screening has to include the detection of human leucocyte antigen (HLA) class I, class II and human neutrophil alloantigen antibodies using established and validated techniques. HLA class I antibody detection should be restricted to antibodies clinically relevant for TRALI. To avoid unnecessary workload, TRALI diagnosis should be assessed by consultation with the reporting clinician and thorough exclusion of transfusion-associated circulatory overload/cardiac insufficiency. In patients diagnosed with TRALI having donors with detectable leucocyte antibodies, evidence of leucocyte incompatibility should be provided by either cross-matching or typing of patient for cognate antigen. CONCLUSION: Leucocyte antibody screening for the immunological clarification of TRALI cases as well as for identification of potentially alloimmunized blood donors is feasible and can be performed in a reasonable and quality assured manner. This practice can contribute to the prevention of antibody-mediated TRALI.

Three novel beta3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency beta3-HPA antibodies.

BACKGROUND: Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging. OBJECTIVES: We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens. METHODS: The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens. RESULTS: Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes. CONCLUSIONS: These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.

The suppressive effect of sphingosine 1-phosphate on monocyte-endothelium adhesion may be mediated by the rearrangement of the endothelial integrins alpha(5)beta(1) and alpha(v)beta(3).

BACKGROUND: Sphingosine 1-phosphate (S1P), known to play important roles in vascular biology, is a bioactive lysophospholipid mediator that maintains endothelial integrity via its cell-surface receptors (S1Ps). In this in vitro study, we aimed to examine the role of S1P in monocyte-endothelium adhesion, which is an important event in the pathophysiology of atherosclerosis. METHODS AND RESULTS: S1P pretreatment of human umbilical vein endothelial cells (ECs), but not U937 cells, effectively suppressed U937-EC adhesion independently from the expression of adhesion molecules, namely ICAM-1, VCAM-1, and E-selectin. This S1P-induced suppressive effect was inhibited by the blockage of S1P(1) and S1P(3) receptors and the specific inhibitors of G(i) protein, Src family proteins, phosphatidylinositol 3-kinase, and Rac1, indicating involvement of these key downstream pathways. Moreover, the RGD peptide and antibodies, which neutralize adhesion via alpha(5)beta(1) and alpha(v)beta(3), effectively inhibited U937-EC adhesion with a degree similar to S1P pretreatment. Both an adhesion assay and flow-cytometric analysis demonstrated that U937 cells adhered through integrins alpha(5)beta(1) and alpha(v)beta(3) expressed on the apical surface of monolayer ECs, and S1P shifted the localization of these integrins from the apical surface to the basal surface. CONCLUSIONS: From the present results, we propose that S1P may contribute to the maintenance of vascular integrity and the regulation of atherogenesis through the rearrangement of endothelial integrins.

A study of dendritic and endothelial cell interactions in colon cancer in a cell line and small mammal model.

AIM: Historically, cancer therapy directly targeting tumor cells have yielded suboptimal clinical results, and therefore anti-angiogenic therapy that targets tumor cells indirectly through impairing tumor vasculature is now considered to be one of the novel approaches potentially effective against various types of cancer. In this study, we evaluated whether lysates of endothelium could be effectively pulsed in dendritic cells (DCs), to enhance their anti-tumor effects. METHODS: For this purpose, we prepared DCs of BALB/c mouse, incubated them with lysates of autologous or xenogeneic endothelium, and tested their anti-tumor effects in two syngeneic models of colon cancer. RESULTS: DCs pulsed with the respective endothelium lysates significantly inhibited the growth of subcutaneous tumors as well as pulmonary metastases in mice, and their anti-tumor effect was superior to that of unpulsed DCs. Immunohistopathological analysis showed significant decrease in the mean vascular density of tumors, correlating well with the extent of tumor inhibition. In vitro analysis of splenocytes isolated from immunized mice revealed an induction of cytotoxic T lymphocytes and activation of natural killer cells, with a lytic activity against activated endothelium but not tumor cells. In addition, antibodies reacting with activated endothelium, but not tumor cells, were detected in murine sera by ELISA, and their function was confirmed by complement-dependent cytotoxicity assay. CONCLUSIONS: Our present results suggest that lysates of endothelium can be effectively pulsed in DCs and enhance their anti-tumor effects through induction of anti-angiogenesis, and therefore should have important clinical implications for adjuvant cancer therapy.

Vaccines targeting tumour angiogenesis--a novel strategy for cancer immunotherapy.

AIMS: To review the concept of tumour angiogenesis and anti-angiogenic therapy, limitations of recently used anti-angiogenic therapeutics; provide an up-to-date overview of the growing number of reports on vaccines targeting tumour angiogenesis; and finally discuss potential complications and future directions in the development of more potent and specific vaccines. METHODS: A literature search was carried out from PubMed for indexed articles. The most important articles were analysed and discussed. FINDINGS: The search yielded a large number of important indexed published articles that were reviewed, screened and tracked for other relevant publications. The most relevant articles, including those previously published by authors, were analysed and discussed. CONCLUSIONS: Recently, different vaccine strategies have been reported to inhibit tumour growth and metastasis by induction of specific cellular and/or humoral immunity against angiogenesis-associated antigens in pre-clinical models, suggesting effective combination of anti-angiogenesis and cancer immunotherapy. Evaluation of tumour endothelial cells and clinical phase I study of the vaccines are recently ongoing, and should give us better insight into the possibilities of this novel strategy for cancer immunotherapy.

Fluctuations of source locations of EEG activity during transition from alertness to sleep in Alzheimer's disease and vascular dementia.

Vascular dementia (VaD) differs from Alzheimer's disease (AD) in larger fluctuations of cognitive impairment, hypothetically because of deteriorated vigilance control. Vigilance levels are reflected by locations of EEG sources. Transition from alertness to sleep might be particularly sensitive to degradations of vigilance control. Twelve AD and 12 VaD patients (medication free, mean age 75.6 and 77.6 years, respectively, difference = n.s.), and 12 healthy elderly subjects (mean age 70.6 years), who served as controls, were studied (each group comprised 2 males and 10 females). A twenty-one-channel EEG was recorded from full alertness to the onset of sleep stage 2. Dipole source modeling, based on Fast Fourier Transform dipole approximation, yielded 3D source localizations in 7 EEG frequency bands. For each brain axis, means of source location differences between successive 20-second periods were calculated (fluctuation magnitude). EEG band-wise MANCOVAs (3 brain axes, 3 subject groups, covariate: age) showed differences in fluctuation magnitude between groups in the 10.5- to 12-Hz alpha(2) frequency band (p=0.0066). Post hoc ANCOVAs for the axes (3 subject groups, covariate: age) were significant on the superior-inferior axis: VaD patients had higher fluctuations than AD patients and controls, without significant difference between the latter two. Thus, larger source fluctuations in VaD might reflect the patients' decreased vigilance control, accounting for their increased fluctuations of cognitive impairment.

Beta-chemokine, macrophage inflammatory protein-1beta (MIP-1beta), is highly expressed in diffuse type human gastric cancers.

Chemokines have been shown to be expressed in some malignant or precancerous tissues. However, the role of these chemokines on tumor development or progression is not clear. The expression patterns of chemokines in gastric cancer tissues were examined in 86 surgically resected samples using immunohistochemistry. Macrophage inflammatory protein-1beta (MIP-1beta) was clearly detected in many gastric carcinoma cells. In most of the differentiated carcinomas, intracellular localization of MIP-1beta was detected in more than 5% of cancer cells, although the percentages of MIP-1beta-positive cells differed among each sample. Undifferentiated carcinomas showed contrasted staining pattern between solid type and non-solid (diffuse) type. MIP-1beta was totally absent in all the poorly differentiated carcinomas with solid type growth pattern (por1). In contrast, MIP-1beta was highly expressed in all of the non-solid type of poorly differentiated carcinoma (por2) and signet-ring cell carcinoma samples. In particular, MIP-1beta was strongly stained in carcinoma cells at the front of invasive lesions. In 43 diffuse type undifferentiated cancers, tumors with high expression of MIP-1beta exhibited significantly more lymph node metastasis. Our results suggest a possibility that MIP-1beta may be related to the scattering and invasion step of gastric carcinoma cells with undifferentiated phenotype.

Prediction of tumor radiosensitivity in rectal carcinoma based on p53 and Ku70 expression.

The identification of predictive indicators of radiosensitivity is extremely useful in selecting patients suited for preoperative radiotherapy and avoiding unnecessary preoperative treatment. In this study, we evaluated the possible role of the immunohistochemical expression pattern of p53 and Ku70 protein in determining tumor radiosensitivity in rectal cancer before preoperative irradiation. We examined pretreatment biopsy materials from 111 patients by immunohistochemistry. The expression pattern of p53 and Ku70 was evaluated for association with tumor radiosensitivity, which was defined according to the criteria of the Japanese Research Society for Cancer of the Colon and Rectum. There was a significant correlation between the expression pattern of p53 and tumor radiosensitivity (P = 0.045); Ku70 and tumor radiosensitivity (P < 0.001); and the combination of p53 and Ku70, and tumor radiosensitivity (P < 0.001). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy in both p53 and Ku70-positive cases for radioresistance were all superior to those of the group positive for p53 alone. In conclusion the examination of the combination of p53 and Ku70 may predict the radiosensitivity of rectal cancer before preoperative irradiation.

Quantification of minimal residual disease in patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia using a real-time RT-PCR assay.

Using a real-time RT-PCR method, we analyzed the expression of e1a2 BCR-ABL mRNA in bone marrow samples from 13 patients with e1a2 BCR-ABL-positive acute lymphoblastic leukemia (ALL) at different time points during chemotherapy and after bone marrow transplantation (BMT). The detection limit of the method, assessed using serial dilutions of ALL/MIK cells, was found to be 1:10(5), similar to what is observed for the conventional RT-nested PCR method. The e1a2 BCR-ABL values were normalized with respect to those of the housekeeping gene GAPDH. The decrease in the e1a2 BCR-ABL/GAPDH ratio after remission induction chemotherapy reflects well the response to chemotherapy and consequently correlates with the prognosis. Although molecular remission was achieved by chemotherapy alone, some patients relapsed, and the e1a2 BCR-ABL/GAPDH ratios in these cases progressively increased to the levels seen prior to hematological relapse. Long-term hematological complete remission (more than 30 months) could be achieved in cases in which a more than 4.0 log decrease in the e1a2 BCR-ABL/GAPDH ratio was obtained by chemotherapy alone, and BMT was then performed. In conclusion, real-time RT-PCR allows for an evaluation of the kinetics of e1a2 BCR-ABL/GAPDH expression during the various phases of chemotherapy or after BMT and may be effective for the indication and control of disease relapse in Ph-positive ALL patients.

Spatial organization of EEG activity from alertness to sleep stage 2 in old and younger subjects.

In order to elucidate brain mechanisms that contribute to the increased tendency for vigilance dysregulation in the elderly, we examined the spatial organization of brain electric activity [electroencephalogram (EEG)] during decreasing vigilance from alertness to onset of sleep stage 2, comparing 7 old and 10 younger, healthy subjects (60-79 and 18-41 years old, respectively). Two features were analyzed: (1) change of location of the brain electric source gravity centers of the EEG frequency bands, and (2) magnitude of fluctuation of these locations over time. Multichannel EEG was analyzed into source gravity center localizations for seven EEG frequency bands, using fast Fourier transform (FFT) Dipole Approximation (first principal component-single source modeling in the frequency domain). Multivariate analysis of covariance (MANCOVA) showed: source localizations were more anterior in old than younger subjects for beta-1 and more superior for all three beta bands; from alertness to sleep, delta and theta EEG sources (inhibitory activity) changed to more posterior and superior areas, and alpha-1 and -2 (routine activity) and beta-1 and -2 sources (excitatory activity) towards anterior and superior areas. Fluctuations of the source locations of delta and beta-2 were larger on the superior--inferior axis, and of beta-2 smaller on the left-right axis in the old than younger subjects. The results suggest functional specifications (inhibitory, routine, excitatory) of cortical positron emission tomography (PET) changes reported in sleep. In sum, aging exhibits changes in spatial organization of EEG-generating neuronal assemblies; during the transition wakefulness-to-sleep, aging affects the spatial-temporal dynamics of this organization. The latter is suggested to contribute to the increased risk for consciousness disturbances in the elderly.

Carcinoembryonic antigen mRNA in abdominal cavity as a useful predictor of peritoneal recurrence of gastric cancer with serosal exposure.

Peritoneal dissemination is the most frequent type of recurrence in patients with gastric cancer with serosal exposure, irrespective of whether they have undergone curative gastrectomy. The purpose of this study was to establish a method to detect micrometastatic cells in the abdominal cavity and predict peritoneal recurrence in patients with such gastric carcinomas. A total of 86 patients with gastric carcinoma, undergoing gastrectomy, were examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect carcinoembryonic antigen (CEA) mRNA in abdominal lavage fluid. Twenty-four cases without serosal exposure were negative, while all 13 cases with macroscopic peritoneal dissemination were positive for CEA mRNA. Among the 49 cases with macroscopic serosal invasion and without peritoneal metastasis, cancer cells were detected in 27 cases with RT-PCR while in only 6 cases with conventional cytology. All cytologically-positive cases were also positive for CEA mRNA. Among the 27 CEA-positive cases, 15 patients (56%) relapsed with peritoneal metastasis within 12 months after gastrectomy. In contrast, none of the 22 CEA-negative cases had peritoneal recurrence within 16-60 months of observation, whereas in 43 cytologically-negative cases, 10 patients relapsed with peritoneal recurrence. As compared with conventional cytological examination, this method would be clinically more beneficial for detecting free cancer cells in the peritoneal cavity and for predicting peritoneal recurrence in gastric carcinoma with serosal invasion.

Peripheral blood dendritic cells, but not monocyte-derived dendritic cells, can augment human NK cell function.

Dendritic cells (DCs) are essential antigen-presenting cells with a wide variety of functions relating to both adaptive and innate immunity. Recently, interactions of DCs with natural killer (NK) cells and NK1.1-positive T cells have been reported in mice. However, in humans, this interaction is not well understood. Here we report the use of a coculture method to analyze the modulation of NK cell function in antitumor immunity by DCs. We found that peripheral blood DCs (PDCs) enhanced NK cell activity in cytotoxicity assay, even without direct contact between DC and NK cells. In contrast, neither monocyte-derived DCs (MoDCs), nor TNF-alpha-treated MoDCs, stimulated NK lytic activity. Secretion of IL-12 and TNF-alpha into the PDC-NK coculture supernatant was increased. However, blocking antibodies against these cytokines could not completely abolish the upregulation of NK activity, suggesting the presence of other soluble factor(s) that affect DC-NK cell interaction. To summarize, this study demonstrates for the first time the direct activation of human NK cells by DC-NK cell interaction in vitro, suggesting that DCs may have a central role linking the innate and adaptive immune responses. Moreover, in stimulating NK cell function, PDCs appear to have a different potential from MoDCs.

Clustered cancer cells show a distinct adhesion behavior from single cell form under physiological shear conditions.

It remains a question whether hematogeneous metastasis arises from a single cancer cell attached to the local endothelium or from a cluster of cancer cells trapped in the vascular bed in the target organ. Adhesive interaction of the single cell form and the clustered form of cancer cells was examined under flow conditions, using two subclones of mouse colon adenocarcinoma Colon 26. A subclone NL17, but not NL14, formed many clusters composed of tumor cells and platelets just after the addition of platelet rich plasma (PRP). Under the shear of 1.0 dyn/cm3, the clustered form of NL17 tethered on laminin or mouse endothelial cell line in hepatic sinusoids (HSE) more frequently than the single cell form of NL17 and NL14. However, all of the clusters showed only transient attachment and never underwent stable arrest on coated laminin, while the single cell form of NL14 and NL17 underwent immediate arrest under shear conditions. On HSE stimulated with TNF-alpha, a small number of NL17 clusters made stable adhesion, although all the clusters detached if the shear stress was increased above 4.0 dyn/cm2. In contrast, the single form of arrested NL17 as well as NL14 remained adherent even at shear of 8.0 dyn/cm2. Compared with single cell, binding of cancer cell clusters to laminin and HSE showed lower resistance to shear stress, although they had adhesive interactions more frequently in flow condition. Since NL17 cells form significantly more metastases by intravenous injection in vivo, our data suggest that "stable adhesion" observed in our flow assay system is not always a prerequisite for clustered cancer cells to develop into metastatic lesions.