Background Activation‐induced protein phosphorylation can be studied by Western blotting, but this method is time consuming and depends on the use of radioactive probes for quantitation. We present a novel assay for the assessment of protein phosphorylation based on latex particles and flow cytometry. Methods This method employs monoclonal antibodies coupled to latex particles to immobilize protein kinase substrates. Their phosphorylation status is assessed by reactivity with phosphoepitope‐specific antibodies. The amount of immobilized protein on the particles was analyzed by direct or indirect immunofluorescence with antibodies to nonphosphorylated epitopes. Results The assay allowed measurement of phosphorylation of multiple protein kinase substrates in stimulated T cells, including the ζ chain of the T‐cell receptor, ZAP‐70, CD3, CD5, SHP‐1, and ERK‐2, using 1–3 μg of total cell protein per sample. The assay provided high resolution of kinetics of phosphorylation and dephosphorylation. Interactions of protein kinase substrates with associated signaling molecules were demonstrated. Conclusions The novel assay allows high‐throughput quantitative measurement of protein modifications during signal transduction. Cytometry 39:250–259, 2000 © 2000 Wiley‐Liss, Inc.

Gastric bezoars are common in cystic fibrosis patients after lung transplantation. The etiology is likely multifactorial, related to gastric motility, respiratory secretions, and medications. Further investigation is needed to understand the pathogenesis of bezoar formation in this selected population, and strategies for primary prevention may be beneficial.

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SummaryThere are no accepted methods to predict the development of platelet transfusion refractoriness (PTR) due to human leucocyte antigen (HLA)-alloimmunization. Hence, matched platelets are usually given only to patients demonstrating PTR, necessarily resulting in some ineffective random donor platelets (RDPLT) transfusions. To assess its utility in predicting PTR, we retrospectively tested samples from 387 patients receiving chemotherapy for acute leukaemia or autologous transplantation using a micro-bead flow cytometry assay. The average of the mean fluorescence intensities (avgMFI) of the class I beads in the screening assay was correlated with outcomes of RDPLT transfusions during a 2 week period. Antibodies were detected in 57 patients; 66 developed PTR, of whom 28 were alloimmunized. avgMFI usefully predicted the development of PTR (area under the receiver operating curve 0Á87, 95% confidence interval: 0Á77-0Á96). A logistic regression model estimated the probability of PTR to be >90% when avgMFI >5440. These results indicate that micro-bead flow cytometry assays could inform a risk-adapted strategy for managing thrombocytopaenic HLA allo-immunized patients.