Since 5-ALA is naturally generated in the human body, it is considered to be relatively safe even after exogenous administration. Indeed, 5-ALA is commercially distributed worldwide as a medicinal drug for photodynamic diagnosis of bladder cancer and malignant glioma and for photodynamic therapy of actinic keratosis and condyloma acuminatum.

Since 5-ALA is metabolized to heme, its use in patients with metabolic diseases of heme synthesis such as acute or chronic porphyria is inappropriate. Hypersensitivity to 5-ALA or porphyrins is also a major contraindication. The liver and kidneys are two of the main accumulation sites for the excretion of 5-ALA; 5-ALA should be used with caution in patients with renal or hepatic insufficiency.

For the detection of LN metastasis in gastrointestinal cancer patients, oral administration of 5-ALA is generally performed [26, 27, 35]. In general, 5-ALA hydrochloride dissolved in water with 50 % glucose is applied orally at a concentration ranging from 15 to 20 mg per kg of body weight. Although the concentration of 5-ALA for oral administration is based on comparison with the detection of primary tumors such as glioma, the optimal administration concentration of 5-ALA for the detection of LN metastasis of various cancers is now under investigation. After the administration of 5-ALA, patients should be protected from strong light such as direct sunlight for 24–48 h to avoid photosensitivity reaction.

Various groups reported that the half-life of 5-ALA in the human body is approximately 45 min, and the maximum concentration of PpIX in blood is reportedly approximately 6–9 h after oral administration of 5-ALA [36–38]. Therefore, in general surgery for gastrointestinal cancer, in which LNs are excised in about 3–5 h after general anesthesia, 5-ALA should be administered to the patients 2–4 h prior to induction of general anesthesia.

The side effects of the administration of 5-ALA have been investigated in patients in several studies [35, 38, 39]. Although substance-specific side effects, such as hypotension, nausea, photosensitivity reaction, and photodermatosis, are reported, these effects are uncommon reactions (less than 1 % incidence). Other side effects such as anemia, thrombocytopenia, leukocytosis, and impairment of liver function have also developed after surgery; however, these seem to be related to procedure involved in the surgery.

Tissues including LNs at several sites at which metastatic lymph nodes are suspected to be present are carefully removed during gastrointestinal surgery. The LNs are isolated from the resected tissues and are analyzed immediately, avoiding unnecessary light exposure. For precise analysis of PpIX fluorescence from the LNs, if necessary, the LNs are cut into a few parts with a fine blade due to the limited penetration depth of excitation light. Fluorescence imaging of the cut surface and diagnostic evaluation of LN metastasis are performed. After the PpIX-fluorescence diagnostic evaluation, the LNs are subjected to a postoperative histopathological diagnosis for precise evaluation of disease.

In this section, we introduce some applications of fluorescence diagnosis of LN metastasis using 5-ALA in human gastrointestinal cancer patients. Firstly, the detection of LN metastasis of gastric cancer is presented.

Figure 28.6 shows representative images of metastatic and nonmetastatic LNs from gastric cancer patients [27]. 5-ALA hydrochloride at 15 mg per kg of body weight was dissolved in 20 ml of 50 % glucose solution and administered orally to the patients 2 h prior to induction of general anesthesia. Isolated lymph nodes were cut in half longitudinally one by one with a fine blade. Fluorescence images of the cut surface were obtained by using a stereomicroscope that comprised a 405-nm band-pass filter for excitation and a color CCD camera equipped with a 430-nm long-pass filter for emission. In practice, the peak wavelength of PpIX fluorescence is about 635 nm, and the fluorescence from PpIX must be filtered with its peak wavelength to obtain the optimum contrast of the PpIX image. Since the PpIX fluorescence observed in the red channel of the color CCD camera is relatively strong compared to the background autofluorescence, the color CCD camera is widely used for the observation of PpIX fluorescence owing to its convenience for use. Furthermore, blue and green fluorescence predominantly represents the distribution of flavins, collagens, and fats, and the analysis of RGB channels of the color CCD camera provides us data for not only the distribution of PpIX but also the distribution of parenchyma and stroma of LNs.

In the metastatic LNs, strong red fluorescence was observed, and the distribution coincided with malignant lesions that were confirmed by HE stain image. In contrast, red fluorescence was weak in nonmetastatic LNs. Results for quantitative analysis of fluorescence intensity of LNs observed on the R channel of the color CCD camera are shown in Fig. 28.7. The fluorescence intensities of the metastatic and nonmetastatic LNs without follicular accumulation are significantly different.

Diagnostic accuracy of the PpIX-fluorescence diagnosis was evaluated with 144 LNs. The PpIX-fluorescence diagnosis was compared with histopathological diagnosis as the gold standard for the detection of LN metastasis. Totals of 17 out of 24 metastatic LNs and 106 out of 120 nonmetastatic LNs were detected by the PpIX-fluorescence diagnostic method with agreement with the histopathological diagnosis. The overall diagnostic accuracy of gross fluorescence inspection was 85.4 % (123/144).

For the PpIX-fluorescence diagnosis, there were false-positive results for 14 nonmetastatic LNs that were deemed to be metastatic and false-negative results for 7 metastatic LNs that were deemed to be nonmetastatic. False-positive results were mainly due to the accumulation of PpIX in normal lymphoid follicles [40, 41]. The normal lymphoid follicles exhibit specific accumulation patterns of PpIX, and the diagnostic specificity and accuracy are, respectively, improved from 88.3 to 96.7 % and from 85.4 to 92.4 % after special consideration of the fluorescence patterns of the malignant lesions and the normal lymphoid follicles. In false-negative results, it is considered that extraneous fluorescence from nonmetastatic regions such as that from collagen, flavins, and fats obscures the PpIX fluorescence from metastatic LNs; in particular, some of the metastatic LNs of patients with scirrhous carcinoma could not be detected owing to strong autofluorescence emitted from increased amount of fibrous tissues. To eliminate the autofluorescence from nonmetastatic regions, detailed analysis of fluorescence spectra of PpIX and nonmetastatic regions is required. One technique of spectral analysis to eliminate autofluorescence of nonmetastatic regions is presented in the next subsection. Individual variation of absorption capacity of 5-ALA may also be in part responsible for false-negative results. Because 5-ALA is mainly absorbed at the upper gastrointestinal tract, patients with diseased stomach may take in 5-ALA of insufficient dose.