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University Medical Center Groningen, Department of Oral and Maxillofacial Surgery, Division of Oncology, The Netherlands
Erasmus Medical Center, Center for Optical Diagnostics and Therapy, Department of Radiation Oncology, Rotterdam, The Netherlands
In vivo measurement of photosensitizer concentrations may optimize clinical photodynamic therapy (PDT). Fluorescence differential path-length spectroscopy (FDPS) is a non-invasive optical technique that has been shown to accurately quantify the concentration of Foscan® in rat liver. As a next step towards clinical translation, the effect of two liposomal formulations of mTHPC, Fospeg® and Foslip®, on FDPS response was investigated. Furthermore, FDPS was evaluated in target organs for head-and-neck PDT. Fifty-four healthy rats were intravenously injected with one of the three formulations of mTHPC at 0.15 mg kg−1. FDPS was performed on liver, tongue, and lip. The mTHPC concentrations estimated using FDPS were correlated with the results of the subsequent harvested and chemically extracted organs. An excellent goodness of fit (R2) between FDPS and extraction was found for all formulations in the liver (R2=0.79). A much lower R2 between FDPS and extraction was found in lip (R2=0.46) and tongue (R2=0.10). The lower performance in lip and in particular tongue was mainly attributed to the more layered anatomical structure, which influences scattering properties and photosensitizer distribution.
Photodynamic therapy (PDT) has been established as a local treatment modality for several kinds of malignancies in various organs.1–7 PDT is based on the use of a light sensitive drug, a photosensitizer, which is locally applied or systemically administered. The photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC or Temoporfin) is one of the most potent clinically used photosensitizers to date.8–10 Its development, study and clinical use was recently summarized in a comprehensive review.11 The formulation of mTHPC in ethanol and propylene glycol, Foscan®, is in use for both curative and palliative treatment of head and neck squamous cell carcinoma (SCC).7,12 The treatment involves excitation of the administered photosensitizer with non-thermal light at the tumor site, which leads to the formation of cytotoxic reactive oxygen species.9,13–17 The amount of reactive oxygen species formed depends on the type of photosensitizer, its concentration, tissue oxygenation, and the rate of irradiation. In head & neck tumors, treatment is typically performed using a mTHPC dose of 0.15 mg kg−1 and light fluence of 20 J cm−2 at a fluence rate of 100 mW cm−2 delivered at 652 nm.11 However, despite the fixed light fluence and administered drug dose differences in PDT response may occur. Monitoring PDT parameters such as oxygenation, light fluence, and photosensitizer concentration during therapy could provide insight in the complex and dynamic interactions that occur during PDT and could give information on the deposited PDT dose.18 Our group recently developed fluorescence differential path-length spectroscopy (FDPS) as a tool to quantify photosensitizer concentration and micro vascular oxygen saturation, a surrogate marker of tissue oxygen concentration.19,20 In previous research, we were able to show that FDPS can be used to measure photosensitizer concentration in vivo in rat liver.21 In this proof-of-concept study, our group used Foscan at 0.3 mg kg−1 as the target photosensitizer. A good linear correlation was found between the mTHPC concentration measured with FDPS and the mTHPC concentration measured by chemical extraction. As a next step towards clinical translation of FDPS for monitoring PDT in head and neck tumors, we here evaluate the performance of FDPS using a clinically relevant drug dose of 0.15 mg kg−1 in target organs for head-and-neck PDT: the lip and the tongue. From a tissue optics point of view it is more challenging to analyze oral mucosal tissues compared to liver tissue. For example, oral tissues such as the dorsum of the tongue and palate are keratinized and are effectively layered media, while the inner lip and floor of the mouth are less so. The keratinisation of the dorsal tongue is present in all mammals, although the degree of keratinisation varies among species.22 In the present study, we have investigated how accurately FDPS measures photosensitizer concentrations in these more optically heterogeneous media. Similar to our previous proof-of-concept study, chemical extraction will serve as the gold standard for mTHPC concentration in these tissues.
One of the problems of Foscan in pre-clinical and clinical PDT is its poor water solubility, which results in aggregation.11,23 Therefore, water soluble liposomal formulations have been designed as nanocarriers for mTHPC. A further advantage of liposomal drug-carrier systems is a reduced uptake by the reticuloendothelial system (RES) and an enhanced permeability and retention effect (EPR).24 Two liposomal mTHPC formulations that have been developed are Foslip® and Fospeg®.8,25–32 In Fospeg, the surfaces of the liposomes are coated by a hydrophilic polymer to further decrease recognition by the RES and thus increase circulation time over Foscan and Foslip.24,33 Both the incorporation of mTHPC into liposomes and the composition of different liposomes are known to significantly influence the spectral properties.28,30 Furthermore, Foslip and Fospeg are known to exhibit different redistribution patterns and liposomal stability in serum.30 We have therefore also investigated the influence of these nanocarriers on FDPS performance.
Fifty-four male Wistar rats (HsdCpb:W) weighing between 250 and 350 g, were purchased from Harlan Netherlands B.V. (Horst, The Netherlands). Three different formulations of mTHPC were kindly provided by Biolitec AG (Jena, Germany); Foscan® (4 mg mL−1 mTHPC), Fospeg® (1.5 mg mL−1 mTHPC) and Foslip® (1.38 mg mL−1 mTHPC). Prior to the experiment, Foscan, Foslip and Fospeg were dissolved for intravenous injection under minimal light and kept at 4 °C in the dark, as recommended by the manufacturer. The dosage used was 0.15 mg kg−1 mTHPC and animals were kept under reduced light conditions (<60 lux). Prior to the experimental measurements the rats were anaesthetized using Isoflurane®/O2/N2O as a general inhalation anesthetic. Variations in mTHPC concentrations are achieved by taking measurements at different time points in the pharmacokinetics profile of each formulation. At 2, 4, 8, 24, 48, or 96 h after injection (n=3 animals per formulation per time point) tissue concentrations of mTHPC were measured using FDPS. In the oral cavity, four measurements were performed on the mucosa of the lip and six on the dorsum of the tongue, all at randomly chosen locations. Next, tissue overlying the liver was dissected which allowed measurements at six randomly chosen locations on the liver. Directly after the optical measurements the animals were terminated by cervical dislocation. Lip, tongue and liver were immediately excised and snap-frozen in liquid nitrogen. FDPS measured the concentration of mTHPC in lip, tongue and liver based on the emitted fluorescence of mTHPC. The concentration estimates determined by FDPS were compared to the concentration determined by chemical extraction. The experimental design for this study was approved by the experimental welfare committee of the University Medical Center Groningen and conformed to Dutch and European regulations for animal experimentation.
The measurement setup used, shown in Fig. 1, was based on the setup described by the group of Amelink et al.19,21 In short, the measurement probe contained two 800 μm fibers at a core-to-core distance of 880 μm. The surface of the probe was polished under an angle of 15 deg to minimize specular reflections during the measurements. One 800 μm fiber, the delivery-and-collection fiber (dc), is coupled to a bifurcated 400 μm fiber, containing a "delivery" and a "collection" leg. The delivery leg is coupled to a 200 μm bifurcated fiber, one leg of which is connected to a xenon light source (HPX-2000, Ocean Optics, Duiven, The Netherlands) and the other leg is connected to a 405 nm diode laser (Power Technology Inc., Arkansas, USA). The collection leg is coupled to another bifurcated 200 μm fiber, of which one leg directly leads to the first channel of spectrograph setup (MC-2000-4-TR2, Ocean Optics, Duiven, The Netherlands), while the other leg leads to a 570 nm long-pass filter before leading into the second channel of the spectrograph. The second 800 μm fiber of the probe, the collection fiber (c), is coupled to a bifurcated 400 μm fiber. One leg is directly coupled to the third channel of the spectrograph, while the other leg leads to the 570 nm long-pass filter, before being coupled in to the fourth channel of the spectrograph.
Schematic diagram of the FDPS measurement setup used in our study. On the right, acquired representative paired DPS spectra and fits (a) and FDPS spectra and fits (b) from the rat lip are shown. The fluorescence spectra demonstrate both autofluorescence and fluorescence attributable to mTHPC.
Before every measurement, the FDPS system was calibrated as described previously.7,19 The measured DPS spectra were fitted to a model extensively described by our group in the literature,20,21,34–36 which returned quantitative estimates of blood volume fraction, micro-vascular blood oxygenation, and vessel diameter. The measured FDPS spectra are corrected for the effect of absorption by multiplying it by the ratio of DPS-signals at the excitation wavelength without and with absorption present, resulting in absorption-corrected FDPS spectra.37 The contribution of mTHPC to the spectra was extracted by using a singular value decomposition (SVD) algorithm38,39 using autofluorescence, Protoporphyrin IX (PpIX), and mTHPC fluorescence as basis spectra.
To determine the concentration of mTHPC in the excised frozen tissues, the chemical extraction method of Kascakova was used40 on small tissue samples (∼0.1 g) of lip, dorsum of the tongue, and liver. In liver it was possible to randomly obtain three samples of liver tissue per animal, representative of tissue located on the liver surface as measured by FDPS. This way, we could average multiple random locations in both optical and chemical concentrations measurements of the liver. In tongue and lip however, we could only obtain one macroscopically representative tissue sample as measured by FDPS, due to the small size of the lip and tongue of rats. All tissue samples obtained were dissolved in 2 mL of the tissue solvent Solvable™ (Perkin Elmer, Groningen, The Netherlands) over 2 h at 50°C with regular stirring. Subsequently, the solubilised solution was diluted further with Solvable™ to an optical density (OD) <0.1. The diluted samples were analyzed in a fluorimeter (Perkin Elmer, Groningen, The Netherlands) by using an excitation wavelength of 423 nm and a spectral detection band of 450 to 800 nm with a resolution of 0.5 nm. The basis spectrum of mTHPC was derived after correction for Solvable™ and autofluorescence components. The concentration of mTHPC was derived from a known calibration curve.40
Confidence intervals on the individual parameters for the individual measurements were determined based on the covariance matrix generated for each fit as described by Amelink et al.41 Differences in fluorescence intensities between formulations and tissue types at similar time points, were determined using one-way ANOVA (two-tailed) with the Bonferroni test for selected pairs of columns. Non-linear regression was used to fit a straight line forced through the origin to characterize the relation between FDPS and chemical extraction for different locations and formulations. To quantify goodness of fit of the regression lines, R2 values and 95% confidence intervals (CI) were determined.
Differences in slope of regression lines between datasets were assessed by the sum-of-squares F-test using a confidence interval of p=0.05. Pearson's correlation coefficient was used (two-tailed, 95% CI) in determining the correlation coefficients (r) between mTHPC fluorescence (FDPS) and blood volume (DPS). Graphpad Prism® (v5.0) was used for all statistical analysis.
Frozen tissue samples of control and mTHPC administered animals were handled under subdued light conditions. Liver, tongue and lip tissue sections of 50 μm were cut and mounted on Starfrost® adhesive glass slides (Menzel, Braunschwig, Germany). Fluorescence images were acquired at 10× magnification using a confocal fluorescence microscope (LSM510, Zeiss, Jena, Germany). Excitation and light collection was performed using a 405 nm laser equipped with a 505 nm long-pass detection filter combined with spectral detection between 545 and 706 nm at 10 nm intervals. Typically 5 μm optical slices were acquired from the center of each 50 μm section. Software written in LabVIEW (v7.1) was used to account for the autofluorescence component of the raw fluorescence; the intensity of resulting images was confirmed to be that attributable to mTHPC fluorescence.
Typical DPS and FDPS spectra and their fits are shown in Fig. 1(a) and 1(b), respectively. The fitted mTHPC contributions of all 54 rats in the lip, tongue and liver at different time points based on the FDPS measurements are shown in Fig. 2(a). The actual mTHPC concentrations determined using chemical extraction are shown in Fig. 2(b).
mTHPC concentration vs. time measured by FDPS (a) and chemical extraction (b) in lip, tongue, and liver tissue for Foscan, Foslip, and Fospeg (error bars indicate SEM). Note the logarithmic scale used for the y-axes
A global comparison of the FDPS and extraction graphs per tissue type in Fig. 2(a) and 2(b), shows a similar trend for both methods as a function of time. One noticeable difference is that FDPS clearly measures more mTHPC in the lip than in the tongue at all time points, whereas the mTHPC concentrations from extraction in these tissue types appear to be very similar. A comparison of mean fluorescence measured by extraction shows no difference (p>0.05) between lip and tongue tissue. However, the same comparison in fluorescence signal measured by FDPS in Fig. 2(a) shows a significantly (p<0.0001) higher intensity fluorescence in the lip.
To further investigate this issue, we plotted the mTHPC component of the FDPS fluorescence versus the mTHPC concentration measured by chemical extraction for each formulation and tissue location within the same rat, as shown in Fig. 3, thereby correcting for possible inter-animal differences in mTHPC uptake and intravenous administration. A linear regression line forced through the origin was used to characterize the relation between FDPS and chemical extraction for different locations and formulations.
Optically measured mTHPC concentration (FDPS) versus true mTHPC concentration (extraction) for 3 different mTHPC formulations (Foscan, Foslip, Fospeg) in lip (a), liver (b), and tongue (c) tissue (error bars indicate SD, logarithmic scales). One measurement point represents multiple FDPS and extraction measurements of 1 rat. Best fit linear regression lines forced through the origin are plotted as solid lines. Pooled data per tissue type (d) show significant differences (p<0.001) in the slopes of the regression lines between the tissue types (linear scales). In (d), for clarity purposes only a portion of the data are shown and error bars are omitted.
In liver tissue, an excellent goodness of fit was found for Foscan, Foslip and Fospeg, as shown in Fig. 3(a). The six regression lines for lip and tongue tissues in Fig. 3(b) and 3(c) showed overall much lower R2, except for Foslip in lip tissue. Pooling of data per tissue-type without discriminating for formulation-type, pictured in Fig. 3(d), clearly showed differences in goodness of fit between different tissue types. In the liver an excellent goodness of fit (R2 of 0.79) was observed, while in lip (R2=0.46) and in particular in tongue (R2=0.10), goodness of fit was much lower.
The influence of mTHPC formulation on FDPS was investigated by assessing differences in slope of regression lines within each tissue type. The sum-of-squares F-test showed a significant (p<0.05, F: 3.252) difference in slope between mTHPC formulations in the liver, with Fospeg showing the highest slope in Fig. 3(a). Similar analysis in lip and tongue tissue, showed no significant (p<0.05) difference in slope between the formulations. Therefore, it is possible to calculate one slope for all three formulations in tongue and lip tissue in Fig. 3(d). In lip and tongue tissue, the goodness of fit to the shared regression line (lip: y=388.6x, 95% CI: 321.1 to 456.2, tongue: y=98.85x, 95% CI: 80.85 to 116.9) remained similar compared to the fit to 3 individual lines. In order to assess the influence of tissue type on the slope regression lines, one overall regression line for liver was computed as well (y=258.4x, 95% CI: 242.8 to 280.5). The differences in slope of the overall regression lines of liver, lip and tongue are clear in Fig. 3(d). Further statistical analysis confirmed the difference visually observed between the regression slopes of lip, tongue and liver: p<0.001, F: 15.70.
To elucidate the differences between the slopes of the regression lines observed for different tissue types, the DPS data were further analyzed for scattering and blood volume fraction differences. Overall analysis of the scattering amplitude per tissue type, shown in Table 1, shows significant differences between tissue types (p<0.05), with the least amount of scattering measured in the liver. No significant difference between the scattering amplitude for different mTHPC formulations was found at any time point in any tissue location. The blood volume fraction was found to have a significant correlation with mTHPC fluorescence only in the liver at the early (2, 4, and 8 h) time points; a significant Pearson's correlation (r) was found with values of 0.90, 0.57, and 0.82, respectively.
To further investigate our findings of a lower correlation between FDPS and chemically extracted samples in lip and tongue versus liver, fluorescence microscopy was performed to determine differences in mTHPC distribution among tissues. Confocal fluorescence microscopy was performed on 50 µm sections of liver, tongue and lip at various time points. Typical examples are shown in Fig. 4. Differences between tissue types are clearly observable; mTHPC is homogeneously spread throughout the liver section, while in lip and especially tongue tissue mTHPC is more heterogeneously distributed. Furthermore, the presence of layered structures can be clearly seen on the transmission images in lip and especially the tongue. In tongue tissue, the filliform papillae can be clearly seen, and these structures do not contain any mTHPC. In lip tissue, a much smaller superficial layer shows no uptake of mTHPC, while an increased uptake is seen in the basement membrane.
Representative confocal fluorescence microscopy of tongue (a), liver (b) and lip (c). Images on the left depict distribution of mTHPC (colored red), corrected for autofluorescence. Images on the right show white light transmission images of the same slide. White scale bar: 500 μm, corresponding approximately with the interrogation depth of FDPS. The arrow in (a) indicates one of the filliform papillae on the surface of the dorsal tongue.
The relationship between mTHPC concentration and therapeutic outcome is complicated as numerous parameters influence the deposited PDT dose. However, the amount of mTHPC present in tissue is clearly an important factor in the PDT dosage. Non-invasive monitoring of mTHPC concentration, as well as other important parameters during PDT, could allow for standardization and optimization of clinical PDT.4,18,42,43 The aim of this study was to test the optical photosensitizer concentration measurement technique FDPS in a more clinically relevant environment compared to previous research performed on liver tissue.21 Therefore, in our present study we used both clinically relevant tissue locations and a clinically used drug dose. Furthermore, we tested the influence of promising new liposomal mTHPC formulations on FDPS performance.
In the liver, linear regression analysis showed an excellent goodness of fit (R2) for the FDPS data to the extraction data, with Foscan, Foslip, and Fospeg showing similar R2. As a further validation for FDPS with our lower drug dose we compared our R2 to the results of Kruijt et al.21 They found a R2 value of 0.87 for Foscan measured by FDPS in the liver; we found a slightly lower R2 value of 0.74. Our R2 values were higher for Fospeg and Foslip at 0.82 and 0.89 respectively. Therefore, our measurements indicate that FDPS results could be reproduced in the liver at the clinically relevant dose of 0.15 mg/kg mTHPC, and extended to the Foslip and Fospeg formulations. Note that although the R2 values can be compared between this study and the study of Kruijt et al., the regression line slopes cannot be compared between these two studies due to differences in the distance between the probe tip and the calibration standard as well as the difference in excitation wavelengths. The shorter wavelength in the current study excites mTHPC at its maximum absorption peak, to maximize mTHPC fluorescence at a lower drug dose. Because both the calibration method and the excitation wavelength were kept constant during our current study, comparison of regression line slopes within our study is possible.
FDPS measurements in a clinically relevant and optically more demanding environment of tongue and lip tissue showed a lower correlation between the FDPS data and the extraction data. Especially in tongue tissue, the correlation was poor with R2 approaching 0 for all formulations. FDPS in lip tissue performed only slightly better. The possible reasons for this poor correlation are discussed below.
The influence of mTHPC formulation on FDPS signal proved to be significant in liver; Fospeg showed a higher slope of the regression line compared to both Foslip and Foscan. This suggests Fospeg has a significant higher quantum yield compared to the other formulations in vivo. This could be explained by a relatively higher amount of non-aggregated mTHPC molecules in liposomal formulations.24 Other in vivo studies also describe a higher fluorescence of Fospeg compared to Foscan,25,26 although in these studies fluorescence intensity is also influenced by formulation specific pharmacokinetics such as aggregation and EPR. The significant difference in slope between the regression lines of Fospeg and Foslip probably depends on the detailed characteristics of the liposomes. It is known that PEGylation of liposomes lengthens the plasma half-life of liposomes, thereby enabling a longer relative monomeric state of mTHPC in Fospeg, and resulting in a relatively higher slope of the regression line compared to Foslip.
In both tongue and lip tissue, no significant difference in slope of the regression lines between the three mTHPC formulations was present. However, this may well be related to the lower goodness of fit and the broader CI of the regression lines in tongue and lip compared to liver, making a significant difference difficult to establish.
Data analysis in Fig. 3(d) shows very distinct differences in FDPS performance by both the goodness of fit (R2) and the slope of the regression lines between tissue types. Fluorescence microscopy showed clear differences in the tissue-specific distribution of mTHPC at all time points; in liver, mTHPC was much more homogeneously distributed compared to both lip and tongue tissue. Furthermore, in tongue tissue an absence of mTHPC fluorescence was seen in the most superficial, dorsal layer around the papillae. In contrast, in lip tissue the distinct basement membrane close to the surface shows more mTHPC fluorescence compared to the stroma. Similar to the tongue, the most superficial layer shows almost no mTHPC fluorescence; however, in lip tissue this superficial layer of the lip is much smaller than in tongue tissue. These differences in distribution of mTHPC can be explained by the known difference in uptake of the dye in various structures like epithelium, lamina propria, striated muscle, smooth muscle, glands and fibro-connective tissue.44–47 While liver tissue consists of multiple similar lobules, lip and tongue have a more complicated, layered composition.
The most important anatomical difference between lip and tongue tissue is the presence of keratinized stratified mucosa in the dorsal tongue while the inner side of the lip is covered by smooth non-keratinized mucosa. Besides tissue specific differences in mTHPC uptake, the biodistribution of mTHPC varies greatly with time.46,48 However, the tissue specific mTHPC distribution is bound to have some influence on optical concentration measurements.
More challenging for our fluorescence measurement are the structural differences between tissue types. The layered, heterogeneous anatomy will certainly influence the tissue specific optical properties, in particular scattering properties. This difference is illustrated by significantly higher scattering amplitudes for lip and tongue tissue compared to liver tissue. Further indication of heterogeneity of lip and tongue tissue is given by the overall larger standard deviations of the scattering amplitude data compared to liver in Table 1.
With knowledge of the microscopic differences observed in anatomy and mTHPC distribution between tissue types, we can explain the tissue specific differences in FDPS performance.
The significant difference we found between correlation coefficients and slopes of the regression lines for different tissue types is potentially caused by a combination of three factors: 1. the layered biodistribution of photosensitizer in combination with the superficial sampling volume of FDPS versus the larger sampling volume of chemical extraction, 2. inter-animal variations in the thickness of the keratin layers, and 3. the large differences in scattering properties between tissue types. Although FDPS yields absorption-corrected data, it does not correct for inter- and intra-tissue scattering differences.19,21,37 As a result, the slope of the correlation between FDPS and extraction will be influenced by the average scattering coefficient of the tissue under investigation. Table 1 shows that the scattering properties vary with tissue type, resulting in different correlation slopes; furthermore, intra- and inter-animal variations in scattering properties are more pronounced in more heterogeneous, layered tissues, such as tongue, resulting in a poorer correlation. A future challenge in improving optical concentration measurement performance would therefore be the ability to correct for scattering.49
With regard to the first two factors, the correlation coefficients and slopes of the regression lines are also affected by a difference in interrogation volume of both techniques (extraction and FDPS). The minimum interrogation volume necessary to obtain accurate extraction data is ∼102 mm3, compared to ∼0.2 mm3 for FDPS. This difference will influence the slope of the regression line in tissue with a relatively heterogeneous, layered mTHPC distribution, as found in tongue and lip tissue. In tongue a large part of the FDPS interrogation volume ∼500 μm of the dorsal tongue consists of filliform papillae, as seen in Fig. 4. Papillae in the rat can be up to 200 μm in length50 and showed decreased mTHPC uptake. Therefore only roughly half the FDPS interrogation volume contains mTHPC, resulting in a lower slope of the regression line between FDPS-extracted mTHPC concentration and chemical extraction in the tongue. Conversely, because the surface of the lip tissue has an increased uptake of mTHPC compared to the surface of the tongue, the FDPS-measured mTHPC fluorescence in the lip increases for the same chemically extracted mTHPC concentration. This explains the significant higher mTHPC fluorescence as measured by FDPS in lip compared to tongue tissue, whereas in the extraction the mTHPC concentrations in these tissue types appear to be very similar. Furthermore, a lower correlation will be found for tissues with more heterogeneous photosensitizer distributions. Although multiple FDPS measurements are averaged for each animal on each tissue type, inter-animal variations in photosensitizer biodistribution with tissue depth will not be averaged out and result in poor correlations. Similarly, inter-animal variations in average keratin layer thickness will also result in poor correlations between the superficially localized FDPS measurement and the "bulk" chemical extraction. The average thickness of the keratin layer in the Wistar rat tongue is described by others as 150 μm (SD±100), measured at a central portion of the dorsal tongue.51 However, the highly keratinized filliform papillae are well known to have substantial, intra-animal morphological variation among differing sites of the dorsal rat tongue.50 This is supported by the even higher variation in average thickness we found for the whole dorsal tongue; 200 μm (SD±120).
Extrapolation of our current results to the clinic is difficult; the dimensions and anatomy in normal human tissue are different compared to that of a rat.52 For example, in humans the keratin layer of normal tissues is on average much smaller than in a rat, which is likely to pose less of a problem for the application of our technique on human tongue.22,52 Furthermore, the pharmacokinetics of mTHPC differs between humans and rodents.23 Another complicating factor is significant spatial variation in mTHPC biodistribution within tumors.48 Moreover, tumors of the oral cavity could also disrupt or change the keratin layer, and therefore influence the performance of our technique. All these aspects may lead to very different observations and very different levels of homogeneity and heterogeneity in human healthy and tumor tissues. In our current pre-clinical study, the emphasis has been on careful investigation of quantitative mTHPC measurements in optically more challenging tissues and of the influence of liposomal formulations. Promising nonetheless were the results of a recent clinical study using FDPS in humans.37 The feasibility of clinical FDPS was shown, as clinical PDT treatments were monitored in three patients with SCCs of the oral cavity.
The non-invasive optical technique FDPS shows promising results in determining the mTHPC concentration in the rat liver for Foscan and for both liposomal formulations; Foslip and Fospeg. In liver, Fospeg showed a significant higher quantum yield compared to the other formulations. In optically homogeneous liver, the correlation with the chemical extraction data was excellent. In the more heterogeneous lip tissue the correlation was lower. In tongue tissue the correlation was poor. The most likely causes of these differences in correlation are the more demanding optical characteristics of lip and especially tongue tissue. In tongue tissue, FDPS performance is even further decreased by a thick layer of keratinized epithelium, which influences the optically sampled mTHPC distribution. Furthermore, in order to accurately monitor mTHPC concentration in heterogeneous tissue, a correction for scattering is needed. This is particularly important for future monitoring of mTHPC in spatially heterogeneous tumor tissues.
Sebastiaan A. H. J. de Visscher ; Max J. H. Witjes ; Slávka Kaščáková ; Henricus J. C. M. Sterenborg ; Dominic J. Robinson, et al."In vivo quantification of photosensitizer concentration using fluorescence differential path-length spectroscopy: influence of photosensitizer formulation and tissue location", J. Biomed. Opt. 17(6), 067001 (Jun 04, 2012). ; http://dx.doi.org/10.1117/1.JBO.17.6.067001
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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
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Display Print Button only on live form.Asked by afmobile on October 09, 2014 at 02:16 AMAnd, last question, can I have a "print" button without a "submit" button? I cannot see a way to provide a "print" button only on the form.
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We can display only the "print" button, it can be done by injecting a custom css code.
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I tried doing it on my end.
1. Navigate to Preferences->Form Styles->Inject Custom CSS
You can use this code:
{ display:none; }
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Chat with Gary Laney
Welcome to GeauxTigerNation! On Tuesday, reporter Gary Laney stops by to chat about the state of LSU football.
Laney joined ESPN.com in 2012 after spending a year and a half as online sports editor of the Baton Rouge Advocate. Before that, he covered LSU sports for the Advocate. He has also been the sports editor at the Beaumont (Texas) Enterprise and covered McNeese State athletics for the Lake Charles (La.) American Press. Laney currently lives in Baton Rouge, La.
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One thing I've been amazed with, in regards to LSU football, is how there always seems to be something to talk about. Doesn't matter that April is about to turn to May, it seems like there is a lot of LSU news to talk about. So let's get started.
Gary Laney (1:59 PM)
And I know links don't work here, but if you paste this in your browser -- http://insider.espn.go.com/blog/colleges/lsu/post?id=9182 -- you'll see my latest LSU 10. Did I get it right? Who's your No. 1 player on the team right now? Would you have included Hill?
Chris Fiegler (Latham,NY)
Out of all the LSU Tigers that got selected in the 2013 NFL Draft,who do you think will be the best in the NFL?
Gary Laney (2:01 PM)
I'm thinking Eric Reid. Love the fit in San Francisco and he can make a quick impact on what's already a good, young team. I can see him doing well for a long time there.
Virginia (Charlotte)
Gary,I am ready to kick Jeremy Hill to the curb for good. I do not care what someone may have said to him. He is a timebomb and lord knows we have had enough of those. Who is coming in as a freshman that could shore up the running game?
Gary Laney (2:03 PM)
Unfortunately for LSU, it did not sign a single running back in the 2013 class. The thought process was to leave the position open for the 2014 class, led by Leonard Fournette. But now with a numbers issue at the position, I'm wondering if LSU wishes it would have taken Adam Taylor, the running back out of Katy, Texas who was interested in LSU -- really tried to commit -- but LSU backed off on him. He wound up signing with Nebraska.
Henry (Montgomery, AL)
Good afternoon Gary,Let's play a hypothetical game. Say LSU loses Hill for the remainder of the season, who are the remaining RBs? Anyone incoming freshmen that may switch to RB?
Gary Laney (2:04 PM)
LSU would have three scholarship running backs: Kenny Hillard, Alfred Blue and Terrence Magee. The best candidate to be switched to RB out of the freshman class is Jeryl Brazil, who was a high school QB who was really good running with the ball.
Henry (Montgomery, AL)
With the off the field troubles that have occurred lately, will Ricky Jefferson even get a chance at LSU?
Gary Laney (2:05 PM)
Jefferson was a sideline guest at the spring game, so I figure he's still in good standing with LSU as of now.
Henry (Montgomery, AL)
Has there been any further word on the status of Leslie? Is he set to be an eligible Tiger this summer?
Gary Laney (2:07 PM)
The last I spoke with people around Leslie was a few weeks ago. He was doing well in school and was on course to finish the semester well and graduate. The class giving him the most trouble is college algebra (I'm sure many can relate). But he was passing the course. Now, it's a matter of waiting until the spring semester is over and seeing if he passed and graduated. If he does, he'll be 100 percent eligible. If not? Then I guess summer school would be next.
coolranch1 (Houston)
The suspension of Jeremy Hill is very disheartening, I was really looking forward to watching him flourish as a fulltime starter.. That being said, if LSU does have to go without Hill for a portion or possibly the entire 2013 season, the backs still on the roster seem more than capable of carrying the load. Thoughts?Also, Do you think this makes it more likely that the coaches put Jeryl Brazil at running back to start his LSU career? Personally I would love to see him as a DB more so than a RB..?
Gary Laney (2:09 PM)
There are those who like Brazil more on offense than CB, where he looks like a natural at first blush, but some feel he might lack the instincts of an elite corner. These folks feel like, on the other hand, his instincts with the ball in his hands are fantastic, so he needs to be on offense. I would be opposed to "starting" Brazil at RB simply to fill the short-term need. If Brazil's future is at DB, he should play DB and find another solution for your fourth RB.
Tony (Richmond, CA)
What would the offseason be at LSU without some off the field drama? Will Jeremy Hill join Tyrann Mathieu as the latest superstar talent Les Miles will have to permanently cut ties with?
Gary Laney (2:11 PM)
It's getting close to that, Tony. LSU did not have to take him after his legal issues in high school. He had to be on a short leash. This has become an issue for LSU -- welcome to the SEC, right? -- and there will be some scrutiny on how LSU deals with Hill.
Jais (ATL)
So what's the deal with bar fights and LSU football players? Geez, this movie is getting old.
Gary Laney (2:13 PM)
Indeed. Of course, old timers will tell you "that's nothing compared to what we did" back in the 50s-70s. But back then, there were no smart phones, social media, etc. Discretion is a big part of the equation now. This isn't the wild west any more. If you get into a bar fight, it's a matter of minutes before social media lets everybody in the world know about it. Didn't these guys get that memo?
Jim (NELA)
What is wrong with the youth these days to keep risk losing so much by continually getting in trouble?
Gary Laney (2:15 PM)
I'm no sociologist, but there is that part of me that thinks that they think it'll be taken care of. We've had several bar fights and at least two recent cases of a recruit and a departing player getting in trouble with police on the street -- not for doing anything terrible, but just for the interaction with the officer. That's a troubling trend.
Ivan (San Francisco)
What is your thought on LSU power house Cardinals?
Gary Laney (2:17 PM)
The Cardinals were smart to pick up defensive guys from LSU, right? I love that Mathieu will play with Peterson, who's sort of the anti-Mathieu. Peterson was always the straight arrow who did everything right on and off the field and he has a big brother relationship with Tyrann. That could work well.
Henry (Montgomery, AL)
Out of all the players that left early, how many were either drafted or signed as free agents?
Gary Laney (2:20 PM)
All 11. 8 were drafted, 3 signed as free agents. I'd say the ones who really were hurt by leaving early were Ford and Faulk, but both have free agent deals.
Sean (Crappy Alabama)
Who do you think wears #7 in the secondary this year?
Gary Laney (2:21 PM)
Wow. Haven't really thought about it. If Loston continues to have a solid summer, I think he might earn it through persistence. He's had injuries and he keeps hanging in there and now he may have a break-out year. That also makes him potential No. 18 material.
Jim (NELA)
Do you see Cam using any special packages this year to get Jennings on the field?
Gary Laney (2:23 PM)
Yes. It's been discussed. I think LSU likes the idea of a mobile QB in the spread option because the matchups it creates. Cameron, as a passing guy, understands what it does to coverages. If you have a talented slot receiver -- and LSU has two potential talented slot guys in Landry and Beckham -- then having a mobile guy who's a threat to run tends to get that guy isolated 1-on-1 with a safety in coverage. It's the reason why LSU recruits safeties who can cover. That's a matchup that everybody is trying to exploit these days.
Henry (Montgomery, AL)
Who wasn't drafted that you felt should have been drafted?
Gary Laney (2:24 PM)
LSU had a good draft. I like Ford's skill set and was a little surprised somebody didn't take him, but I won't go so far as to say he should have been drafted.
Ben (Richmond, KY)
Who were you most surprised to see go undrafted? Faulk was a projected 1st rounder before his injury (wish the Saints would have taken him in a late round); Ford had a phenomenal workout at the combine, I thought for sure some team would fall in love with his skills; and Wing was rated by nearly every service as the top punter.
Gary Laney (2:26 PM)
Sort of repeat question, but I'll elaborate on your specific asks. Faulk never looked "right" to me. I don't think he was healthy enough. I agree on Ford. I don't think it matters much for a punter like Wing. Kicker/punters aren't usually draft guys. I know another punter (at least 1) was picked, but I think Wing was in better position not being picked so he could negotiate to the team with the biggest need.
Les Miles (Baton Rouge, LA)
Hey Gary, do you think Leonard Fournette can reclassify like they do in basketball, and suit up for me this fall? :)
Gary Laney (2:27 PM)
If that were true, first play of the Cam Cameron OC era would be toss lead up the middle with the freshman running back ... and it would be a good call.
Derrin (Plano, TX)
How much did Tharold Simon's brush with law impact his draft stock?
Gary Laney (2:29 PM)
Not the least bit at first blush. The third-round talk I heard early on sounded overly optimistic. He looked like a mid-late round guy and he went in the fifth round. Sounds about right. Now, would he have gone higher had he not been arrested? I can't say that with confidence.
Scott (Los Angeles)
I think I brought up off-field issues last chat and what they can do to curb them. Well, after Hill's arrest I now put the blame on the coaches. To some degree it's the players' own choices, but three years in a row of some sort of arrest/suspension shows the coaches aren't making any impressions. It's gone from inconvenient to embarrassing. How hard is it to convince these players that nothing that happens off the field or campus is worth endangering their eligibility and possible career?
Gary Laney (2:31 PM)
I tend to agree. I've always been of the opinion that any one action is accountable to the individual involved but trends are accountable to culture and leadership. Jeremy Hill's arrest, if the charges prove true, would be Jeremy Hill's fault. But several off-field issues in relatively short order reflect leadership and team culture which starts with coaches.
Anthony (Tampa, FL)
Do you see any chance of Hill getting reinstated onto the team before the start of the season if found not guilty?
Gary Laney (2:32 PM)
If he's cleared of charges, he'll be reinstated faster than the judge can bang his gavel.
Henry (Montgomery, AL)
How much do you think Sam Montgomery's comments hurt him in the draft? Looking at his production and numbers, he far exceeded those of Mingo yet...
Gary Laney (2:35 PM)
I think it hurt him a little. You're right about his production but he's a guy whose production allowed you to overlook some apparent flaws. He's not the quickest guy off the ball, for example (though he's hardly slow). I think his words allowed the focus to move from what he does well to what his flaws were. If he were a 100-percent, can't-miss, slam-dunk (Clowney-like), he could have said he tends to drift off to sleep just before the snap and he still would have not been hurt.
Ivan (San Francisco)
With so many LSU players in NFL, its will getting harder to keep up with all of them. I hope they leave a mark in the league. Which NFL team with LSU players do you think will be among the best next year?
Gary Laney (2:36 PM)
You're wanting me to say Niners now that Eric Reid is their No. 1 pick, don't you Ivan from San Francisco? And yeah, the Niners are my answer. Super Bowl team. Young. I'm hoping Lattimore can get healthy in a year or two, so when the tread on Frank Gore's tires wear out, he'll be ready to step in.
Kevin (Houston)
I know there have been a lot Hill questions but I don't think anyone has straight up asked you if you think he'll be on the team come fall?
Gary Laney (2:38 PM)
My gut says yes. From what I can see, he punched a guy in a bar fight. I think there may be penance for this -- even in light of past transgressions -- that likely won't involve permanent dismissal. The key is getting to quick resolution so he can get to whatever penance that is.
Anthony (Tampa, FL)
Are there any top recruits close to committing right now?
Gary Laney (2:40 PM)
Don't know if you call him a "top" recruit, but I think defensive end Deondre Clark has to be close. So is tight end Anthony Jones. Right now, it seems like the top guys on LSU's board are wanting to go through the process, take visits, etc. I can't think of an ESPN 150 guy who I expect to commit before summer.
Ivan (San Francisco)
Do you feel there is a drop off in LSU special teams in 2012 after McGaughey replaced Joe Robinson?
Gary Laney (2:42 PM)
I thought LSU continued to be dominant in special teams. Remember, McGaughey was the guy in charge in 2011 and that was one of the great special teams seasons in LSU history, wasn't it?
Scott (Los Angeles)
I saw you think Loston is No. 18 material. I think they will give it to Anthony Johnson. But is 7 becoming a secondary prize, literally? Matheiu wanted it because of PP, but there's no lineage before that is there?
Gary Laney (2:43 PM)
The importance of "7" only goes back to Peterson. You are correct. Could be Mills too, right? I like Mills at corner and "7" has gone to cornerbacks up to now. Another potential 18 could be Barrow. Or Blue coming off the injury. Lots of guys.
Mike the Tiger (BR)
Who hasn't gotten into a fight or some sort of troubles at Reggie's? No biggie, next story.
Gary Laney (2:45 PM)
Isn't that why Reggie's is there? For LSU students to get into fights? But Mike, here's the question: How many of these guys are on probation?
Sean (Crappy Alabama)
I feel like a lot of people are counting LSU out this year because of all the players we lost. Yet I see all the players coming in and the existing players we have, and I think we could be dangerous.... What do you think ?
Gary Laney (2:45 PM)
Discounting LSU is a bad idea because the consistency of talent LSU attracts and its track record in getting young talent prepared to play quickly.
Henry (Montgomery, AL)
Of this upcoming year's juniors and seniors, who looks like a potential first round draft pick?
Gary Laney (2:46 PM)
Johnson, Collins, possibly Barrow (might be a reach). Hill if he comes back, stays out of trouble and has a big year.
Ben (Richmond, KY)
I heard a rumor that LSU is in discussions with Wisconsin about playing a game at Lambeau Field for the 2014 season. I really like the idea and hope it happens, but they say it's unlikely if the SEC switches to a 9 conference game schedule. Have you heard anything lately on the possibility of playing 9 conference games in the future?
Gary Laney (2:48 PM)
It's been reported, so it's not just a rumor. LSU does like a big early game to help create urgency in August preps. LSU was a really focused team early in 2011 because the opener against Oregon. It's part of the reason you see TCU on the schedule this year. The 9-game schedule will be discussed at the SEC spring meetings next month. I wouldn't say it's likely, but it'll be a key discussion.
Ivan (San Francisco)
I think next upgrade to LSU football facilities is sports bar with extra security, which will be the only authorized option if players want to go to bars :)
Gary Laney (2:49 PM)
Nothing could possibly go wrong with that idea... :)
Henry (Montgomery, AL)
After the spring game, did any recruits move LSU to the top of their board?
Gary Laney (2:51 PM)
I think LSU left that weekend in very good shape with ILB Clifton Garrett, who raved on LSU after coming in for the spring game weekend. I don't know if he has called LSU his leader, but it just looks like he's loving some LSU right now.
treyTrey (Florida) [via mobile]
Is lsu on the outside looking in for jebrill peppers now?
Gary Laney (2:52 PM)
I'd say yes. He didn't get blown away from Baton Rouge and left still a Michigan lean, best I can tell. LSU is probably unlikely for him.
Michael (Picayune)
Is bama in the lead for Cam robison?
Gary Laney (2:55 PM)
It's close right now. LSU offered Ronnie Clark, an Alabama ESPN 150 safety who is originally from Robinson's home town of Monroe and knows Robinson well. Recent LSU commit Garrett Brumfield is friends with Robinson. Those things help LSU. I think the depth of quality in Louisiana will help LSU with in-state prospects. The more Louisiana guys continue to commit to LSU, the more their peers may want to be part of it too.
Henry (Montgomery, AL)
After seeing the spring game, where do you think LSU SHOULD (not will) be ranked in the preseason?
Gary Laney (2:57 PM)
Somewhere between 10-15, I'd say. I think LSU is potentially better than that, but there are two issues: 1. Schedule, and 2. There is talent that needs to prove it. For example, I think LSU has a dominant end, maybe 2, on its roster. But somebody is going to have to prove it on Saturdays because I give the Tigers credit for having one.
Jim (NELA)
How much better will the offense have to be this year to help compensate for all of the lost players on defense?
Gary Laney (2:59 PM)
Significantly better. I think the big-picture strategy might be tweaked. In recent seasons, the apporach has been to play defense and field position, meaning the offense might be conservative. This year, the tendency might be to be aggressive offensively, especially early. That's a significant burden for the offense to have to carry.
Gary Laney (2:59 PM)
Significantly better. I think the big-picture strategy might be tweaked. In recent seasons, the apporach has been to play defense and field position, meaning the offense might be conservative. This year, the tendency might be to be aggressive offensively, especially early. That's a significant burden for the offense to have to carry.
Michael (Picayune)
Do you think lamin, anthony johnson, lofton and ego will be high draft picks this next year?
Gary Laney (3:00 PM)
Loston, you mean. I think the last two have work to do on their stock. The first two I think have a great chance.
Henry (Montgomery, AL)
Has anyone gauged where LSU sits on Dylan Moses board? Never too early right?
Gary Laney (3:01 PM)
He's already committed to LSU .... at least LSU's on-campus high school, University Lab. Dad's a fan, I think. That gives LSU a nice edge.
Michael (Picayune)
How is LSU looking with Hootie Jones?
Gary Laney (3:02 PM)
Work to do. Alabama is still there with them. Right now, rule of thumb is that LSU has work to do with Monroe guys and, with some exception, is in pretty good shape with I-10 guys.
Dave (Lagos, Nigeria)
What are your thoughts on Mickey Johnson getting more playing time and in the mix at DT with AJ and Ego
Gary Laney (3:03 PM)
Work to do. Lots of quality freshmen will be pushing Mickey and Quentin Thomas for a spot on the 2-deep. But Johnson had a solid spring. He has some physical limits -- he is simply short and not very long -- but Mickey's solid.
Rob (Texas)
Could LSU's class of 2014 could be best recruting class ever at LSU?
Gary Laney (3:03 PM)
If they get most of the top guys on the board within 200 miles of campus, it would definitely be in the discussion.
Michael (Picayune)
If Zach has a stellar year could he be a first rounder?
Gary Laney (3:04 PM)
He'll be a draft guy I suspect. A first-rounder? That would take a lot of work, especially with the NFL trending even more toward mobile QBs.
Gary Laney (3:04 PM)
He'll be a draft guy I suspect. A first-rounder? That would take a lot of work, especially with the NFL trending even more toward mobile QBs.
Glen (New Orleans)
Do you think Cam Cameron will allow a RB to have a season like Charles Scott had? We never give our backs a chance to gain 1,000 yards and I think in the end it always hurts us.
Gary Laney (3:05 PM)
Glen, you are forgetting Ridley, who dominated the carries in his one season as starter and put up big numbers. I think if Hill is cleared, he'll start the year as the featured back and will remain that unless he gets banged up or is ineffective.
Gary Laney (3:07 PM)
Guys. Great chat, but running late. There are some leftover questions I'll get to in Friday's mailbag. If you have any more questions, email me at @email@example.com or tweet me your question at @glaneyespn and I'll get them into Friday's mailbags. And I'll be back, same time same place next Tuesday!
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To discover the real players, the people who drive American domestic and foreign policy and make all of the important decisions, you only need to focus on a hand full of people…
Submitted by Michael Krieger, Liberty Blitzkrieg:
I never liked the saying: "We are the 99%." While admittedly catchy and effective as a slogan, I think it is ultimately divisive and counterproductive. The reason I say this is because the statement itself alienates much needed allies for no good reason.
In a country with a population of 320 million, the 1% represents 3.2 million people, which is a pretty big number. While the 1% certainly have far superior material lives compared to the 99%, that doesn't mean a particularly large percentage of them are thieves, cronies or oligarchs. In fact, it behooves people interested in transitioning to another paradigm to court as many of them as possible to the cause. It is very useful to have well meaning people with resources and connections on your side. To blithely assume there aren't plenty of potential allies from a pool of 3.2 million is committing strategic suicide.
– From the post: Charting the American Oligarchy – How 0.01% of the Population Contributes 42% of All Campaign Cash
Much of my focus throughout 2015 was on the pernicious influence of the 0.01%, i.e., the American oligarchy. Indeed, nothing would please oligarchs more than to define a struggle as the 99% vs. the 1% in order to shift attention away from the real root of the problem, themselves.
As I've mentioned time and time again, 99% of the 1% doesn't bribe politicians, write tax laws, or influence U.S. foreign policy. To discover the real players, the people who drive American domestic and foreign policy and make all of the important decisions, you only need to focus on a hand full of people.
Today, the New York Times published an important article that proves the point. Here are the key paragraphs in the entire lengthy article:
The impact on their own fortunes has been stark. Two decades ago, when Bill Clinton was elected president, the 400 highest-earning taxpayers in America paid nearly 27 percent of their income in federal taxes, according to I.R.S. data. By 2012, when President Obama was re-elected, that figure had fallen to less than 17 percent, which is just slightly more than the typical family making $100,000 annually, when payroll taxes are included for both groups.
From Mr. Obama's inauguration through the end of 2012, federal income tax rates on individuals did not change (excluding payroll taxes). But the highest-earning one-thousandth of Americans went from paying an average of 20.9 percent to 17.6 percent. By contrast, the top 1 percent, excluding the very wealthy, went from paying just under 24 percent on average to just over that level.
This is merely a reflection of what I've been saying throughout…


text:
(Washington, DC – April 20, 2015) The United States Department of Justice (DOJ), the Federal Bureau of Investigation (FBI), the Innocence Project and the National Association of Criminal Defense Lawyers (NACDL) reported today that the FBI has concluded that the examiners' testimony in at least 90 percent of trial transcripts the Bureau analyzed as part of its Microscopic Hair Comparison Analysis Review contained erroneous statements. Twenty-six of twenty-eight FBI agent/analysts provided either testimony with erroneous statements or submitted laboratory reports with erroneous statements. The review focuses on cases worked prior to 2000, when mitochondrial DNA testing on hair became routine at the FBI. The DOJ, FBI, Innocence Project and NACDL have been working jointly on this review and share the same goal of ensuring the integrity of the American justice system in all respects. All of the parties are committed to addressing this situation properly and will continue to work together in a collaborative and professional manner."The Department has been working together with the Innocence Project and NACDL to address errors made in statements by FBI Examiners regarding microscopic hair analysis in the context of testimony and laboratory reports. Such statements are no longer being made by the FBI, and the FBI is also now employing mitochondrial DNA hair analysis in addition to microscopic analysis," said Amy Hess, Executive Assistant Director, Science and Technology Branch, FBI. "However, the Department and the FBI are committed to ensuring that affected defendants are notified of past errors and that justice is done in every instance. The Department and the FBI also are committed to ensuring the accuracy of future hair analysis testimony, as well as the application of all disciplines of forensic science. The Department and FBI have devoted considerable resources to this effort and will continue to do so until all of the identified hair cases are addressed.""These findings confirm that FBI microscopic hair analysts committed widespread, systematic error, grossly exaggerating the significance of their data under oath with the consequence of unfairly bolstering the prosecutions' case," said Peter Neufeld, Co-Director of the Innocence Project, which is affiliated with Cardozo School of Law. "While the FBI and DOJ are to be commended for bringing these errors to light and notifying many of the people adversely affected, this epic miscarriage of justice calls for a rigorous review to determine how this started almost four decades ago and why it took so long to come to light. We also need lawmakers in Washington to step up and demand research and national standards to prevent the exaggeration of results in reports and in testimony by crime lab analysts."Norman L. Reimer, Executor Director of NACDL added, "It will be many months before we can know how many people were wrongly convicted based on this flawed evidence, but it seems certain that there will be many whose liberty was deprived and lives destroyed by prosecutorial reliance on this flawed, albeit highly persuasive evidence. Just as we need lawmakers to prevent future systemic failures, we need courts to give those who were impacted by this evidence a second look at their convictions."The FBI and DOJ agreed to conduct a review of criminal cases involving microscopic hair analysis after the exoneration of three men convicted at least in part because of testimony by three different FBI hair examiners whose testimony was scientifically flawed. The Innocence Project and NACDL, with its partners David Koropp, Partner at Winston & Strawn LLP, and his colleagues, and Michael R. Bromwich, Managing Principal of The Bromwich Group, who served as the Inspector General of DOJ from 1994-1999, worked with the FBI and DOJ in determining the scope and protocols for the review. The review encompasses cases where FBI microscopic hair comparison was used to link a defendant to a crime and covers cases in both federal and state court systems. It does not, however, cover cases where hair comparison was conducted by state and local crime labs, whose examiners may have been trained by the FBI. The FBI has trained hundreds of state hair examiners in annual two-week training courses.The government identified nearly 3,000 cases in which FBI examiners may have submitted reports or testified in trials using microscopic hair analysis. As of March 2015, the FBI had reviewed approximately 500 cases. The majority of these cases were trials and the transcript of examiner testimony was reviewed. Some of these cases ended in guilty pleas, limiting the review to the original lab report. In the 268 cases where examiners provided testimony used to inculpate a defendant at trial, erroneous statements were made in 257 - 96% of the cases. Defendants in at least 35 of these cases received the death penalty and errors were identified in 33 (94%) of those cases. Nine of these defendants have already been executed and five died of other causes while on death row. The states with capital cases included Arizona, California, Florida, Indiana, Missouri, Ohio, Oklahoma, Pennsylvania, Tennessee and Texas. It should be noted that this is an ongoing process and that the numbers referenced above will change.All but two of 28 FBI examiners provided testimony that contained erroneous statements or authored lab reports with such statements. The review has shown that the FBI Examiners testified in cases in 41 states.In light of these findings, the Department of Justice and FBI have committed to working with the Innocence Project and NACDL to take the following steps:
- Conduct an independent investigation of the FBI Laboratory protocols, practices and procedures to determine how this occurred and why it was allowed to continue for so long.
- Continue aggressive measures and review the process to determine whether additional steps could be taken to secure the transcripts and/or lab reports and review the hundreds of remaining cases that may contain invalid scientific statements.
- Strongly encourage the states again to conduct their own independent reviews where its examiners were trained by the FBI.The Innocence Project, NACDL and Winston & Strawn LLP are assisting the Department of Justice as it works to locate and notify defense counsel of the results of this review - especially critical in the cases of each person where error was identified in accordance with the protocols established for the review. NACDL is working to ensure that all individuals who were defendants in affected cases will have access to a volunteer lawyer to review this new evidence, advise them on how it may impact their conviction, and challenge convictions based on the invalid evidence in appropriate cases. The legal groups are not releasing the names of the defendants affected at this time, leaving it to the defendants and their lawyers to determine what to do with the information and whether to disclose the error to the press.The FBI has agreed to provide free DNA testing where there is either a court order or a request for testing by the prosecution. Additionally, in federal cases, DOJ will not raise procedural objections, such as statute of limitations and procedural default claims, in response to defendants' petitions seeking a new, fair trial because of the faulty evidence. But the majority of the FBI examiner testimony was provided in state court prosecutions, and it will be up to the individual states to determine if they will follow DOJ's leading in permitting these cases to be litigated.Before mitochondrial DNA testing was used to analyze hair in criminal cases, prosecutors throughout the country routinely relied on microscopic hair comparison to link a criminal defendant to a crime. The practice was deemed "highly unreliable" in the 2009 National Academy of Sciences report on forensic science, Strengthening Forensic Science in the United States: A Path Forward. Nevertheless, some jurisdictions continue to use hair analysis where mitochondrial DNA testing is deemed too expensive, time consuming or is otherwise unavailable. According to Innocence Project data, 74 of the 329 wrongful convictions overturned by DNA evidence involved faulty hair evidence.Over the course of 25 years, the FBI conducted multiple two-week training courses that reached several hundred state and local hair examiners throughout the country and that incorporated some of the same scientifically flawed language that the FBI's examiners had used in some lab reports and often in trial testimony. In response to the FBI/DOJ review, the Texas Forensic Science Commission has already begun a review of cases handled by analysts at state and local crime labs. Similar audits are needed in most other states.
Monday, April 20, 2015
NACDL press release: FBI Testimony on Microscopic Hair Analysis Contained Errors in at least 90% of Cases in Ongoing Review
Wednesday, April 8, 2015
Selective Silence of Defendant Cannot Be Used in Prosecution's Case In Chief
by James Eckert, Monroe County Assistant Public Defender
The Court of Appeals, Fahey, J. writing for the court, held: "as a matter of state evidentiary law, that evidence of a defendant's selective silence generally may not be used by the People as part of their case-in-chief, either to allow the jury to infer the defendant's admission of guilt or to impeach the credibility of the defendant's version of events when the defendant has not testified." (People v Williams, 2015 NY Slip Op 02866 [4/7/15]) Thus, if a defendant waives Miranda, speaks to police about rape accusations, but refused to answer whether he had sex with the complainant, his refusal to answer could not be used to imply consciousness of guilt (the prosecution said the defendant "did not deny" having sex with the complainant) or to cast doubt on his other statements about the incident. "A defendant who agrees to speak to the police but refuses to answer certain questions may have the same legitimate or innocent reasons for refusing to answer as a defendant who refuses to speak to the police at all." The defendant's DNA was found in the complainant, he testified at the Grand Jury that sex was consensual, but did not testify at trial.
While this is merely an extension of the general rule that a defendant's silence cannot be used against him, it is important to have it clarified. The court noted that there are unusual circumstances in which silence will be admissible (People v Rothschild, 35 NY2d 355 [1974] [defendant police officer had a duty to report to his supervisors if he was taking the bribe money as part of a "sting"]; People v Savage, 50 NY2d 673 [1980] [defendant told police he shot victim during an altercation, properly cross-examined on his failure to make claim that victim was trying to rob him, as he testified at trial]).
The primary difference was that those two cases involved cross-examination, not introduction of the defendant's silence as part of the prosecution's case in chief, "In those cases, the People used conspicuous omissions from the defendants' statements to police during cross-examination of the defendants, in order to impeach the credibility of the exculpatory testimony provided by the defendants at trial. Here, by contrast, the People introduced, as part of their case-in-chief, evidence regarding defendant's failure to tell the detective during custodial interrogation that he and the victim had consensual sex." The prosecution claimed that they were, effectively, cross-examining the defendant's grand jury testimony. However, as the court pointed out, the prosecution chose to introduce this testimony itself. "The People may not introduce evidence that they deem favorable to defendant on their direct case and impeach that evidence, also on their direct case, with evidence of defendant's silence." The court also noted that they introduced the selective silence before the grand jury testimony.
Therefore, the rule in New York is that "Evidence of a defendant's selective silence therefore generally may not be used by the People during their case-in-chief and may be used only as 'a device for impeachment' of a defendant's trial testimony in limited and unusual circumstances".
Two judges would have held the error harmless.
Monday, April 6, 2015
Police Officers May Not Testify That They They Believe the Defendant is Guilty
Evidence as to what was said by an interrogating police officer during the interrogation, including testimony that officer told the suspect/defendant that he thought he was lying in denying committing the crime is arguably admissible as it tended to explain to the jury the circumstances of the alleged statements, thereby countering a claim that the statement was not voluntarily obtained (People v Walden, 148 AD2d 971 [4th Dept 1989]). However, in People v Pabon (--- AD3d ----, 2015 WL 1380113 [4th Dept 3/27/15]), the Appellate Division, Fourth Department, citing People v Kozlowski (11 NY3d 223 [2008]) has recently held that it is error for the prosecutor to elicit that, in fact, it was the officer's opinion that the defendant lied during the interrogation when he denied committing the charged crimes or to explain why he believed that the defendant way lying.
In Kozlowski, the Court of Appeals explained what a witness may and may not say with respect to an opinion as to whether the defendant's version of events is credible. (People v Kozlowski, 11 NY3d 223 [2008]). Witnesses are permitted to testify about facts, but not as to their opinion as to whether the defendant's version of events was believable:
The line is crossed not when a witness relates facts that may be prejudicial, but when he or she conveys-either directly or indirectly-a personal opinion regarding the defendant's criminal guilt (citations omitted).... What was impermissible about the testimony was that its sole purpose was to bolster the testimony of another witness by explaining that his version of the events was more "believable" (citation omitted). It was thus the equivalent of an opinion that the defendant was guilty, which is impermissible.
(People v Kozlowski, 11 NY3d 223, 240 [2008].)
Police officer testimony that they believed that defendant lying in denying guilt is precisely of the type that the Court of Appeals described as improper. This is not a recent or novel rule. Previously, the Court held that where, as here, a prosecutor seeks testimony that the defendant's version of events was not believable, the witness's ". . . testimony is equivalent to an opinion that the defendant is guilty, and the receipt of such testimony may not be condoned (People v Williams, 6 NY2d 18, 23 [1959]; People v Higgins, 5 NY2d 607, 627-628 [1959]; People v Gradon, 43 AD2d 842 [2d Dept 1984])." (People v Ciaccio, 47 NY2d 431, 439 [1979].)
Police officer testimony that they believed that defendant lying in denying guilt is precisely of the type that the Court of Appeals described as improper. This is not a recent or novel rule. Previously, the Court held that where, as here, a prosecutor seeks testimony that the defendant's version of events was not believable, the witness's ". . . testimony is equivalent to an opinion that the defendant is guilty, and the receipt of such testimony may not be condoned (People v Williams, 6 NY2d 18, 23 [1959]; People v Higgins, 5 NY2d 607, 627-628 [1959]; People v Gradon, 43 AD2d 842 [2d Dept 1984])." (People v Ciaccio, 47 NY2d 431, 439 [1979].)
In People v Glover (195 AD2d 999 [4th Dept 1993]), the Court held that it was not improper for a police officer to describe what he had told the suspect prior to the suspect making a statement. The Court held that the officer's statement to the suspect of the evidence of guilt ". . . was not hearsay because the testimony was not offered for its truth, but to establish the circumstances in which the statement was obtained, and to rebut defendant's argument that the officer coerced or fabricated defendant's statement." People v Glover, 195 AD2d 999 (4th Dept 1993). By contrast, what is prohibited is for a prosecutor to elicit from the officer the officer's opinion that the defendant was lying when he denied responsibility. That is the very type of testimony which this Court implied in Glover would be improper and should be precluded as it tends to usurp the factfinder's function (People v Jennings, 33 AD3d 378 [1st Dept 2006]).
This rule parallels the rule which prohibits a prosecutor from expressing her personal belief to the jury that the defendant is a liar. (People v Grice, 100 AD2d 419, 421 [4th Dept. 1984] [prosecutor's remarks improperly placed before the jury the prosecutor's personal belief that the defendant was lying]; People v Shanis, 36 NY2d 697, 699 [1975]).
Just as a prosecutor exceeds the bounds of legitimate advocacy by expressing, as the prosecutor did in this case, a personal opinion on a defendant's truthfulness (see People v Wlasiuk, 32 AD3d 674 [2006], lv dismissed 7 NY3d 871[2006]), she may not elicit the police officer's opinions on a defendant's truthfulness or guilt.
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