Dual-channel red/blue fluorescence dosimetry with broadband reflectance spectroscopic correction measures protoporphyrin IX production during photodynamic therapy of actinic keratosis

Abstract

Dosimetry for aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy of actinic keratosis was examined with an optimized fluorescence dosimeter to measure PpIX during treatment. While insufficient PpIX generation may be an indicator of incomplete response, there exists no standardized method to quantitate PpIX production at depths in the skin during clinical treatments. In this study, a spectrometer-based point probe dosimeter system was used to sample PpIX fluorescence from superficial (blue wavelength excitation) and deeper (red wavelength excitation) tissue layers. Broadband white light spectroscopy (WLS) was used to monitor aspects of vascular physiology and inform a correction of fluorescence for the background optical properties. Measurements in tissue phantoms showed accurate recovery of blood volume fraction and reduced scattering coefficient from WLS, and a linear response of PpIX fluorescence versus concentration down to 1.95 and 250 nM for blue and red excitations, respectively. A pilot clinical study of 19 patients receiving 1-h ALA incubation before treatment showed high intrinsic variance in PpIX fluorescence with a standard deviation/mean ratio of > 0.9. PpIX fluorescence was significantly higher in patients reporting higher pain levels on a visual analog scale. These pilot data suggest that patient-specific PpIX quantitation may predict outcome response.

Figures

Fig. 1

(a) Schematic of the multichannel dosimeter setup showing system components. (b) Fiber arrangement of the patient-interface (probe tip), which is used in light compression contact-mode. The source fiber is represented in blue. The surrounding six fibers transmit light remitted from the tissue through the filter wheel and into the spectrometer. Photographs of the internal optical assembly and the chassis are shown in (c) and (d), respectively.

Fig. 2

Fluorescence measurements in protoporphyrin IX (PpIX) dilution series as measured with blue excitation light. Phantoms were constructed with Intralipid 1%, whole blood 1%, and Tween 5%. (a) A representative spectral fit is shown for [PpIX]=500  nM. Sixteen dilutions were sampled between 4000 nm and 0.1 nM. The fitted PpIX fluorescence versus known PpIX concentration is shown (b) on a linear scale and (c) on a log scale with the lower limit of detection identified at 1.95 nM. The integrated fluorescence area versus PpIX concentration is shown in (d) with the lower limit of detection identified at 250 nM.

Fig. 3

Fluorescence measurements in PpIX dilution series as measured with red excitation light. Phantoms were constructed with Intralipid 1%, whole blood 1%, and Tween 5%. (a) A representative spectral fit is shown for [PpIX]=1000  nM. Sixteen dilutions were sampled between 4000 nm and 0.1 nM. The fitted PpIX fluorescence versus known PpIX concentration is shown (b) on a linear scale and (c) on a log scale with the lower limit of detection identified at 250 nM. The integrated fluorescence area versus PpIX concentration is shown in (d) with the lower limit of detection identified at 1000 nM.

Fig. 4

(a) Representative white light spectra sampled in tissue phantoms for Intralipid 1% and blood volume of 1%. Estimated versus known values for (b) reduced scattering coefficient at blue and red excitation wavelengths and (c) blood volume fraction. Error bars on (b) and (c) represent variation between phantoms.

Fig. 5

(a) and (b) The residual error between fitted PpIX fluorescence emission spectra excited by blue excitation light for uncorrected and corrected spectra, respectively. (c) and (d) The residual error between fitted PpIX fluorescence emission spectra excited by red excitation light. Residual is calculated as the percentage deviation for fitted PpIX fluorescence normalized to average of estimates from all phantoms.

Fig. 6

Visual analog scale (VAS) pain as reported by patients immediately after photodynamic therapy (PDT) treatment. # indicates that pain was not assessed on ID1.

Fig. 7

Representative optical measurements of fluorescence emission with excitation with (top) blue, (middle) red, and (bottom) white light reflectance from patient ID1. Left-hand panels show measurements 1 h following aminolevulinic acid administration (pre-Tx). Right-hand panels show measurements immediately after administration of treatment light (post-Tx).

Fig. 8

PpIX fluorescence measured in response to blue excitation. (a) Patient-specific fitted PpIX fluorescence pre- and post-Tx. Box-plots show comparison of (b) absolute PDT dose and (c) percent PpIX photobleaching with patient reporting low pain (VAS<5) and high pain (VAS>=5).

Fig. 9

PpIX fluorescence measured in response to red excitation. (a) Patient-specific fitted PpIX fluorescence pre- and post-Tx. Box-plots show comparison of (b) absolute PDT dose and (c) percent PpIX photobleaching with patient reporting low pain (VAS<5) and high pain (VAS>=5).

Fig. 10

Patient-specific optical parameters measured before and after PpIX PDT treatment: (a) blood volume fraction, (b) microvascular saturation, (c) reduced scattering coefficient at 637 nm, and (d) volume fraction of melanocytes in the epidermis.

Fig. 11

Patient-specific ratio of red to blue PpIX fluorescence; * marks indicate zero PpIX. Box-plot shows comparison of ratio with patient reporting low pain (VAS<5) and high pain (VAS>=5).

Fig. 12

Monte Carlo simulation data showing influence of PpIX depth distribution on the sampled depth of fluorescence origin and the ratio of collected light intensities. Three distributions were considered: (a) uniform PpIX throughout, (b) superficial PpIX, and (c) deep PpIX. The 80% fluorescence sampling depth is reported for blue and red excitation channels, and the red/blue ratio is given for each case.