Effects of temperature and anticoagulant on the in vitro quantitation of Leukocyre Expressed Mac-1 and Post-traumatic assay to predict the development of ARDS, /

Abstract

Adult Respiratory Distress Syndrome (ARDS) is post-traumatic disease state in whch the lungs are compromised, resulting in aberrant function. The objective of this study is to determine if there are differences in expression of the cell adhesion molecule Mac-1 on whte blood cells for ARDS patients compared to patients who do not develop ARDS. Control studies first examined the effect of anticoagulant and incubation temperature on the in vitro expression of Mac-1. Blood fiom healthy volunteers was collected into tubes containing either EDTA, heparin or oxalate and incubated at room temperature for 0, 5, 10, 15, 30 and 60 minutes before being placed on ice (prevents in vitro stimulation). Subsequently, the leukocytes were indirectly labeled with fluorescence conjugated antibodies, isolated and then analyzed by flow cytometry. Based on the control study results it was determined that incubating blood samples at room temperature in EDTA, followed by placing the sample on ice w i t h 15 minutes would produce minimal effects on Mac-1 expression in vitro. Therefore EDTA was chosen as the best anticoagulant for patient studies. Patient studies involved collecting blood fiom trauma patients in EDTA containing tubes at 0, 6, 12, 18, and 24 hours after initial trauma. The patient analysis utilized a protocol similar to the control stules. ARDS patients were found to show increased Mac-1 expression in EDTA anticoagulated blood when compared to non-ARDS patients. Therefore, these studies suggest that Mac-1 is differentially expressed in post-traumatic patients who will develop ARDS when compared to patients who do not develop ARDS.