Lower Body Ischemia-Reperfusion during: METHODS

Institutional human subjects committee approval and individual informed consent were obtained prior to the study. Eight Caucasian male patients aged 55-81 with past history of hypertension, tobacco smoking and arteriosclerotic cardiovascular disease undergoing elective infrarenal abdominal aortic aneurysm repair were studied. Radial artery and pul- induction of general anesthesia, a right femoral vein monary artery catheters were placed prior to induecatheter was placed. Each patient received 80-100 tion of general endotracheal anesthesia. After the    units per kilogram of heparin intravenously 10 minutes before application of the aortic clamp. Mean aortic cross-clamp time was 85 minutes (range 47-130 minutes), and mean duration of surgery was 241 minutes (range 150-315 minutes). There were no intraoperative complications.

Blood Collection

Blood from the radial artery catheter was collected before general anesthesia; 30 minutes after the skin incision; 30 minutes after placement of the aortic clamp; and at one-, five-, 10-, 15-, 20-, 25-, 30-, 40-, 50-, 60- and 90 minutes after release of the aortic clamp. Blood was collected from the femoral vein catheter at intervals identical to the arterial samples, except that no femoral vein blood was collected before general anesthesia. The samples were collected into plastic tubes containing EDTA (Bec-ton-Dickinson, Rutherford, NJ).
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Monoclonal Antibodies

The monoclonal antibodies (mAb) used in the study were antihuman mouse IgG, conjugated with either phycoerythrin (PE) or fluorescein isothiocyanate (FITC) (CDllb-PE, CD18-FITC, Becton-Dickinson Immunocytometry Systems, San Jose, CA).

Leukocyte Preparation

One-hundred-milliliter aliquots of arterial blood were immediately incubated with 10 ml of the CD1 lb and CD 18 mAbs for 15 minutes at room temperature in the dark. The erythrocytes were then lysed using FACS lysing solution (Becton-Dickinson Immunocytometry Systems, San Jose, CA). The leukocytes were then separated by centrifugation at 500Xg for five minutes and washed twice with phosphate buffered saline (PBS) containing 0.1% sodium azide. The cells were fixed in 0.5% paraformaldehyde in PBS for 30 minutes at 4°C, washed twice with PBS, stored at 4°C and analyzed within 24 hours.
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Flow cytometric Analysis

A minimum of 10,000 leukocytes per sample was analyzed using a FACSort flow cytometer and LYSYS-II computer software (Becton-Dickinson Immunocytometry Systems, San Jose, CA). Each cell was illuminated by an argon laser (488 nm), and three parameters recorded for each: forward light scatter (FSC), side scatter (SSC), and fluorescence intensity for either phycoerythrin (580 nm) or fluorescein isothiocyanate (530 nm), as appropriate. Data for neutrophils and monocytes were extracted and analyzed separately by gating on these populations using morphological characteristics displayed on a dot plot of FSC versus SSC.

Fluorescence intensity was recorded using a four-decade, 1,024-channel logarithmic scale. Histograms of frequency versus fluorescence channel number were derived for each cell type, allowing calculation of mean fluorescence channel (MFC), which correlates directly with antigen expression. MFC was corrected for autofluorescence and nonantigen-specific antibody binding by using standard isotype control monoclonal antibodies.

Complete Blood Counts

Complete blood counts were performed on aliquots of each sample using a routine automated procedure.
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Data Presentation and Statistical Methods

All data are presented as mean ± standard deviation. Mean fluorescence of CDllb and CD 18 are expressed for each leukocyte population in each sample. Multiple-measure ANOVA was performed to evaluate the significance of changes in the parameters measured over different time points and paired t-tests were done to compare arterial vs. venous data. P values <0.05 were considered statistically significant.