Extending fluorescent microsphere methods for regional organ blood flow to 13 simultaneous colors

Abstract

Seven fluorescent microsphere colors can be used in a single experiment to estimate regional blood flow without correcting for spillover of emitted fluorescence. To extend the method to 13 colors, we compared the accuracy of three methods for spillover correction. Fixed wavelength intensities were corrected by matrix inversion, and synchronous scan spectra were corrected by least squares fit of an overdetermined system of linear equations and by least squares fit of a sum of Gaussian and Lorentzian functions. Correction methods were validated in pigs and sheep by simultaneous injections of radioactive microspheres and fluorescent microspheres of 7, 10, and 13 different colors. We induced extreme changes in flow to create regions with low fluorescent signals bound on either side by high fluorescent signals. Blood flow was determined by radioactivity and by fluorescence using both fixed excitation and emission wavelength pairs and synchronous scanning and then corrected for spillover. Correlation between fluorescent intensity and radioactivity were excellent for all three correction methods [R2 = 0.98 +/- 0.02 (mean +/- SD)]. Low-flow regions requiring large spillover correction had systematic errors for some color combinations in all methods. We conclude that for 13 fluorescent colors spillover error can be minimized so that all three correction methods provide accurate estimates of regional blood flow.

Microsphere maps of regional blood flow and regional ventilation

“…The availability of 13 different fluorescent labels has substantially expanded investigational opportunities for simultaneous measurements of both regional perfusion and regional ventilation with the small tissue samples (51). The correlation for simultaneously administered different FMS colors uniformly exceeds 0.99 for the standard ϳ2-cm 3 lung piece size, and these measurements correlate equally well with simultaneous measurements made with RMS (18).…”

Gas Exchange and Pulmonary Hypertension following Acute Pulmonary Thromboembolism: Has the Emperor Got Some New Clothes Yet?

“…19 The simultaneous measurement of regional V and Q in small regions of the embolized lung was first accomplished more than a decade ago, by investigators who used a technique based on aerosolized and injected fluorescent microspheres (FMSs). [20][21][22] Their data made it possible to reconstruct all regions of the embolized lung post mortem, with each region designated for its own V/Q ratio 23 and mapped in three-dimensional (3D) coordinates. All emboli locations were also marked.…”

Repeat Microsphere Delivery for Serial Measurement of Regional Blood Perfusion in the Chronically Instrumented, Conscious Canine

“…Eight fluorescent microsphere colors can be used in a single experiment to estimate RBF without correcting for spillover of emitted fluorescence. It has been reported that for 13 fluorescent colors, spillover error can be minimized so that all 13 colors provide accurate estimations of RBF [25]. In large animal studies, investigators often inject 6 to 18 million spheres during a single injection [8][9][10][11][12][13].…”

“…We demonstrated that eight serial injections totaling 18 million spheres administered over an extended protocol are feasible without concern for a statistically or substantively significant influence on RBF from excessive accumulation of microspheres in the capillaries after multiple, large injections. Therefore, using the 13-color technique [25], it may be possible to measure RBF in the same chronic preparation up to 13 times, provided that the cumulative injection of microspheres is less than 18 million spheres.…”