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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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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<0.01) between phagocytic index and titer of HLA class I antibody measured by lymphocyte immunofluorescence test-flow cytometry. In addition, the phagocytic index obtained by FMPA with pHrodo-SE was significantly higher than that obtained by FMPA with the previously used dye, carboxyfluorescein diacetate succinimidyl ester, when platelets were sensitized by w6/32 and anti-HLA class I antibody-containing sera. Because of the higher resolution and higher sensitivity than those of the previous method, the pHrodo-SE-based FMPA may be suitable for more precise quantitation of phagocytosis activity, which would enable qualitative evaluation of transfusion effectiveness., (Copyright © 2018.)

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<0.01) between phagocytic index and titer of HLA class I antibody measured by lymphocyte immunofluorescence test-flow cytometry. In addition, the phagocytic index obtained by FMPA with pHrodo-SE was significantly higher than that obtained by FMPA with the previously used dye, carboxyfluorescein diacetate succinimidyl ester, when platelets were sensitized by w6/32 and anti-HLA class I antibody-containing sera. Because of the higher resolution and higher sensitivity than those of the previous method, the pHrodo-SE-based FMPA may be suitable for more precise quantitation of phagocytosis activity, which would enable qualitative evaluation of transfusion effectiveness.

The differences in the interaction of monoglycerides with bile acids or cholesterol have been investigated from a physico-chemical point of view. A Langmuir trough and fluorescence microscope was used to study mixed monolayers of DL-α-palmitin (a monoglyceride) and cholesterol or deoxycholic acid (a bile acid) at the air/aqueous interface. The surface pressure-area per molecule isotherms of the monolayers were analyzed to give the thermodynamic properties. The deoxycholic acid-DL-α-palmitin monolayer showed stronger repulsions between the film components than was observed with the cholesterol acid-DL-α-palmitin monolayer. Mixed monolayers containing DL-α-palmitin and cholesterol or deoxycholic acid phase separated at high surface pressures and high fractions of DL-α-palmitin, the conditions that resulted in the most repulsions between the two components of the monolayer. The mixed cholesterol and DL-α-palmitin monolayer phase separated in a random pattern. The deoxycholic acid and DL-α-palmitin mixed monolayer gave smaller domains that were distributed in a homogeneous fashion within the monolayer at high molecular packing densities. The difference in the cholesterol and deoxycholic acid interactions with DL-α-palmitin were explained by the fact that while both cholesterol and deoxycholic acid molecules do not pack efficiently with the DL-α-palmitin molecules, the attractive interactions between the alcohol groups on DL-α-palmitin and the carboxylic groups on deoxycholic acid cause attractive interactions between the deoxycholic acid and DL-α-palmitin domains, which causes the interaction abilities of deoxycholic acid with DL-α-palmitin to be higher than cholesterol., (Copyright © 2014 Elsevier B.V. All rights reserved.)