USE OF AN AUTOMATED FLUORESCENT MICROSPHERE METHOD TO MEASURE REGIONAL BLOOD-FLOW IN THE FETAL LAMB

We have developed a method for measuring regional blood flow by means of fluorescent microspheres in all organs and tissues of the fetal lam b, including brain, heart, lung, liver, gut, spleen, kidney, adrenal, brown fat, skin, muscle, bone, and placenta. Five different fluorescen t-labeled microspheres were used: blue (B), yellow-green (Y), orange ( O), red (R), and crimson (C). An automated, 96-well microplate fluores cent reader (bottom reading) was chosen for the assay because of the r apidity and high throughput that it offers. Tissue samples were digest ed by 4 M ethanolic KOH. The sedimentation method and dye extraction w ith Cellosolve acetate, as previously reported by others, were used fo r the sample processing. The bones were crushed and allowed to directl y soak in Cellosolve acetate to extract the dye. The relationship betw een microsphere number and fluorescent intensity was linear over a bro ad range of microsphere numbers (80 - 20 000/mL). The coefficients of variation of within-run and between-run precision were 3.39 +/- 1.10% and 4.54 +/- 1.10%, respectively. Recovery of microspheres from tissue s and blood averaged 94.3 +/- 2.5% and was not dependent on microspher e number. The spillover of the fluorescent signals into adjacent color s was 4.0 +/- 0.1% for O to Y, 8.1 +/- 0.4% for O to R, and 9.1 +/- 0. 5% for R to C, and these values were constant over a wide range in con centrations of the microsphere pairs. No evidence was obtained for que nching of the emission of one fluorophore via photon absorption by ano ther fluorophore. The measurements of regional blood flow obtained wit h fluorescent microspheres in three chronically instrumented fetal lam bs at similar to 140 days gestation were similar to the flow estimates obtained using radioactive microspheres in four other fetal lambs at the same gestational age. The fluorescent method is thus a viable alte rnative to the radioactive technique for the measurement of regional b lood flow to all fetal organs and tissues, particularly when an automa ted fluorescent microplate reader is employed to reduce analysis time.