BACKGROUND Multiparameter DNA flow cytometry using a one‐laser bench‐top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four‐color analysis. Therefore, we optimized and extended our three‐color method (Corver et al., 1994, Corver et al. 1996) to a four‐color analysis of phenotypic intra‐tumor heterogeneity using a bench‐top flow cytometer. METHODS First, the effect of a range of different propidium iodide (PI) and TO‐PRO‐3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde‐fixed lysolecithin‐permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells. Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti‐keratin antibodies. Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3‐W vs. FL3‐A). The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity. Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell‐adhesion molecule (Ep‐CAM; APC fluorescence), (b) keratin (R‐phycoerythrin [RPE] fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein‐isothiocyanate [FITC] fluorescence) to label stromal cells. RESULTS Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50–100 μM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25–2.0 μM. Average CVs were 2.58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter‐ or intra‐DNA stem line heterogeneity of Ep‐CAM expression was observed in the keratin‐positive fractions. Vimentin‐positive, keratin‐negative cells were restricted to the DNA diploid fraction. CONCLUSIONS PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four‐color high‐resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench‐top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories. Cytometry 39:96–107, 2000 © 2000 Wiley‐Liss, Inc.
BackgroundIn ambulatory lower limb surgery, spinal anesthesia with rapid onset and a short duration of block is preferable. We hypothesized that the use of 2-chloroprocaine would be associated with a faster motor block recovery compared with prilocaine in knee arthroscopy. A difference of 15 min was considered clinically relevant.Methods150 patients were randomly allocated to receive intrathecally either 40 mg of 2-chloroprocaine or 40 mg of prilocaine. The primary outcome was the time to complete recovery from motor blockade. Secondary outcomes included time to full regression of sensory block, peak sensory block level, urine retention needing catheterization, time until hospital discharge, incidence of transient neurologic symptoms and patient satisfaction.ResultsTime to complete recovery from motor blockade was 15 min shorter for 2-chloroprocaine (median: 60 min; IQR: 60–82.5) than for prilocaine (median: 75 min; IQR: 60–90; p=0.004). 2-Chloroprocaine also resulted in faster full regression of sensory block (median: 120 min; IQR: 90–135 compared with median: 165 min; IQR: 135–190, p<0.001) and faster time to hospital discharge (mean difference: 57 min; 95% CI 38 to 77, p<0.001). Peak sensory block was higher in the 2-chloroprocaine group (median: T9; IQR: T6–T12 compared with median: T10; IQR: T8–T12, p<0.008). Patient satisfaction and urine retention needing catheterization were equal in both groups.ConclusionsIn knee arthroscopy, spinal anesthesia with 2-chloroprocaine results in a faster recovery of motor and sensory block, leading to quicker hospital discharge compared with prilocaine.Trial registration numberNTR6796.
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