Raman spectroscopy of bladder tissue in the presence of 5-aminolevulinic acid.

Raman spectroscopy has the ability to provide differential diagnosis of different cancers with high sensitivity and specificity. A major limitation in its clinical application is the weak nature of Raman signal, which inhibits scanning large surface areas of tissues. In bladder cancer diagnosis, fluorescence-guided endoscopy with 5-aminolevulinic acid (5-ALA) has gained interest as a technique that can provide such spatial differentiation, thus improving early detection and more complete removal of superficial tumors. However, several studies have demonstrated the poor specificity of this modality. Combining fluorescence with Raman spectroscopy could improve its diagnostic capability. However, little is known about the effect of agents such as 5-ALA on Raman spectra of tissue. In this paper, we present measuring Raman spectroscopy from benign and malignant bladder tissues in the presence of 5-ALA and attempt to evaluate the potential to discriminate between different pathologies. Raman spectra were recorded from 92 bladder biopsies without 5-ALA and 38 biopsies with 5-ALA using a Raman microspectrometer system at 830nm excitation. Empirical and multivariate statistical techniques were used for data analysis. Algorithms were developed to determine the effect of 5-ALA on tissue and its influence on the prediction ability of a preliminary benign/malignant prediction model. In samples with 5-ALA, an overall decrease in Raman intensity was observed when compared to the Raman spectra from samples without 5-ALA. Additionally, differences in relative intensities at 1270 and 1330cm(-1) were also noted. However, significant differences were observed in the Raman spectra of benign and malignant samples with 5-ALA indicating the potential of using Raman spectroscopy for discriminating bladder cancer in the presence of 5-ALA. The Principal-Component fed Linear-Discriminant Analysis (PCA/LDA) algorithm derived from biopsies in the absence of 5-ALA used to predict biopsies in the presence of 5-ALA resulted in an overall sensitivity and specificity of 42.6% and 71.1%, respectively. This suggests the presence of 5-ALA in tissue affects the Raman spectra. A PCA/LDA algorithm based on fluorescence information (i.e. PpIX fluorescence positive or negative) and the Raman spectrum of 5-ALA biopsies, had a sensitivity and specificity of 100% and 80.8%, respectively. This study demonstrates that applying 5-ALA affects the Raman spectra of bladder tissues. However, benign/malignant differentiation can be accomplished with a preliminary PCA/LDA algorithm, suggesting the potential of a combined diagnostic modality in vivo.

Assessment of fiberoptic near-infrared raman spectroscopy for diagnosis of bladder and prostate cancer.

OBJECTIVES: To determine whether a fiberoptic Raman system, suitable for in vivo use, is able to differentiate between benign and malignant bladder and prostate pathologic findings in vitro. Raman spectroscopy is an optical technique that provides a measure of the molecular composition of tissue by analyzing the way that tissue scatters laser light. Laboratory studies have shown that the technique can be used to identify and characterize transitional cell carcinoma and prostate adenocarcinoma in vitro. METHODS: A total of 220 Raman spectra were recorded from 29 snap-frozen bladder samples collected at cystoscopic procedures, and 197 Raman spectra were recorded from 38 snap-frozen prostate samples collected at transurethral resection of the prostate. The spectra were correlated with the histologic features and used to construct separate diagnostic algorithms for the bladder and prostate. These algorithms were tested as to their ability to determine the pathologic finding of a sample from its Raman spectrum. RESULTS: The bladder algorithm was able to differentiate benign samples (normal and cystitis) from malignant samples (transitional cell carcinoma), with an overall accuracy of 84%. The prostate algorithm was able to differentiate benign samples (benign prostatic hyperplasia and prostatitis) from malignant samples (prostate cancer), with an overall accuracy of 86%. CONCLUSIONS: The results of this study have demonstrated that the clinical Raman system can provide an accurate and objective method to diagnose prostate and bladder cancer in vitro. Because the Raman probe is suitable for use during endoscopic, laparoscopic, or open procedures, this work paves the way for in vivo studies.

The use of Raman spectroscopy to identify and characterize transitional cell carcinoma in vitro.

OBJECTIVE: To determine whether Raman spectroscopy can be used to differentiate between normal, inflammatory and malignant bladder pathologies in vitro, and secondly if it can used to grade and stage transitional cell carcinoma (TCC). MATERIALS AND METHODS: In all, 1525 Raman spectra were measured from 75 bladder samples comprising normal bladder, cystitis, carcinoma in situ (CIS), TCC and adenocarcinoma. Multivariate analysis was applied to the spectral dataset to construct diagnostic algorithms; these were then tested for their ability to determine the histological diagnosis of each sample from its Raman spectrum. RESULTS: The diagnostic algorithms could be used to accurately differentiate among the pathological groups, in particular, a three-group algorithm differentiated among normal bladder, cystitis and TCC/CIS with sensitivities and specificities of > 90%. Algorithms could also accurately characterize TCC in terms of splitting them into low (G1/G2) or high (G3) grade and superficial (pTa) or invasive (pT1/pT2) stage. CONCLUSION: Raman spectroscopy can be used to accurately identify and grade/stage TCC in vitro. The technique therefore shows promise for use as an objective method to assist the pathologist in assessing bladder pathologies. Raman spectroscopy also has potential to provide immediate pathological diagnoses during surgical procedures. Following the promising results of this in vitro study, in vivo cystoscopic studies are planned.

The use of Raman spectroscopy to identify and grade prostatic adenocarcinoma in vitro.

Raman spectroscopy is an optical technique, which provides a measure of the molecular composition of tissue. Raman spectra were recorded in vitro from both benign and malignant prostate biopsies, and used to construct a diagnostic algorithm. The algorithm was able to correctly identify each pathological group studied with an overall accuracy of 89%. The technique shows promise as a method for objectively grading prostate cancer.

The significance of cagA(+) Helicobacter pylori in reflux oesophagitis.

BACKGROUND: Helicobacter pylori is a gastroduodenal pathogen associated with ulceration, dyspepsia, and adenocarcinoma. Recent preliminary studies have suggested that H pylori may be protective for oesophageal adenocarcinoma. In addition, strains of H pylori identified by the presence of the cytotoxin associated gene A (cagA) are shown to have a significant inverse association with oesophageal adenocarcinoma. Given that cagA(+) H pylori may protect against oesophageal carcinoma, these strains may be protective for oesophagitis, a precursor of oesophageal carcinoma. AIMS: The aim of this study was to investigate the association between cagA(+) H pylori and endoscopically proved oesophagitis. PATIENTS: The study group included 1486 patients attending for routine upper gastrointestinal tract endoscopy. METHODS: At endoscopy the oesophagus was assessed for evidence of reflux disease and graded according to standard protocols. Culture and histology of gastric biopsy specimens determined H pylori status. The prevalence of cagA was identified by an antibody specific ELISA (Viva Diagnostika, Germany). RESULTS: H pylori was present in 663/1485 (45%) patients and in 120/312 (38%) patients with oesophagitis. Anti-CagA antibody was found in 499/640 (78%) H pylori positive patients. Similarly, anti-CagA antibody was found in 422/521 (81%) patients with a normal oesophagus and in 42/60 (70%) with mild, 24/35 (69%) with moderate, and 11/24 (46%) with severe oesophagitis. The risk of severe oesophagitis was significantly decreased for patients infected with cagA(+) H pylori after correction for confounding variables (odds ratio 0.57, 95% confidence interval 0.41-0.80; p=0.001). CONCLUSIONS: These results suggest that infection by cagA(+) H pylori may be protective for oesophageal disease.

A comparison of six commercial kits for Helicobacter pylori detection.

The British Society of Gastroenterologists suggests that dyspeptic patients under 45 years of age should be screened serologically for Helicobacter pylori infection, to reduce endoscopy workload. We have compared the sensitivity, specificity, and predictive value of six commercial serological kits intended for pre-endoscopy screening for H. pylori with histopathology and culture in 82 dyspeptic patients, 35 of whom were H. pylori positive. The kits' sensitivities were as follows: Bio-Rad GAP 100%, Helico-G 100%, Premier 97%, and Pyloriset EIA-G 94%. Poor specificity of the ELISA kits--Bio-Rad GAP 67%, Helico-G 67%, Premier 85%, and Pyloriset EIA-G 76%--was due to previous treated or cleared H. pylori infection. Allowing for previously documented H. pylori infection or peptic ulcer improved specificity--Bio-Rad GAP 84%, Helico-G 84%, Premier 100%, and Pyloriset EIA-G 90%. The Pyloriset Dry latex kit had a higher specificity (86%) but a lower sensitivity (75%) than the Oxoid latex kit (specificity 70%, sensitivity 94%). The qualitative Premier Launch kit had the best overall results (and was the easiest ELISA to perform). Reliable serological diagnosis of H. pylori is now suitable for screening dyspeptic patients.

Preservation of gastric antral mucus is associated with failure of eradication of Helicobacter pylori by bismuth, metronidazole and tetracycline.

METHODS: Forty-three patients positive for Helicobacter pylori by histology and culture of antral biopsies (n = 40) or histology alone (n = 3) were investigated. They received either regimen 1--tripotassium dicitrato bismuthate 120 mg q.d.s. and tetracycline 250 mg q.d.s. for 4 weeks, with metronidazole 200 mg q.d.s. for the first 2 weeks, or regimen 2--omeprazole 20 mg b.d., amoxycillin 500 mg t.d.s., tetracycline 500 mg q.d.s. each for 3 weeks. Gastric antral biopsies were scored (0-3) histologically for mucus depletion, polymorphonuclear and mononuclear cell infiltrate. H. pylori eradication was assessed by biopsy and culture 1 month after the cessation of treatment. RESULTS: With regimen 1, pre-treatment mucus depletion was significantly higher where eradication was successful (median score 2) compared to where it was not (median score 1, P < 0.01); there were no differences in the scores for polymorphonuclear or mononuclear cell infiltrates. In patients receiving regimen 2, there were no differences in either mucus depletion or polymorphonuclear or mononuclear cell infiltrate, between those where eradication was successful and those where it was not. Metronidazole minimum inhibitory concentrations rose when eradication with regimen 1 was unsuccessful (median before 0.19 mg/L, median after treatment 16 mg/L; P = 0.04). CONCLUSION: Pre-treatment mucus depletion is identified as a factor affecting H. pylori eradication. Preservation of mucus may facilitate acquisition of metronidazole resistance.

Long-term follow up of patients with gastritis associated with Helicobacter pylori infection.

The aim of this study was to evaluate the long-term prognosis for patients suffering from gastritis associated with Helicobacter pylori infection, and in particular the proportion of cases progressing to peptic ulcer. The study was carried out in one urban general practice. One hundred and three patients who had presented with dyspepsia over the 1973-80 period and who were found to have a macroscopically normal endoscopy were reassessed between seven and 14 years later. Gastric antral biopsies had been taken routinely at endoscopy and were subsequently re-examined for the presence of H pylori. The patients' medical records were examined to establish their consulting rates over the follow-up period and whether they suffered from any other medical conditions. Patients were interviewed to assess the course of their dyspeptic symptoms. Comparison of patients who were unequivocally H pylori positive with those who were negative revealed no significant differences in the consultation rate for gastroenterological symptoms, in the proportion of patients referred to a hospital consultant or for further gastroenterological investigations or in the proportion reporting that their symptoms had improved. However, a statistically highly significant relationship was found between H pylori infection and hypertension. The results of this study have shown that there is a good prognosis for non-ulcer dyspepsia whether or not H pylori infection is present. The association between H pylori gastritis and hypertension clearly merits further investigation.

Detection of Campylobacter pylori by the biopsy urease test: an assessment in 1445 patients.

The presence of C pylori infection was determined in 1445 patients undergoing upper gastrointestinal endoscopy over a 12 month period. The presence of C pylori was detected in gastric mucosal biopsy specimens by the biopsy urease test, microscopy (Gram stained smears and histology) and culture. Two media were used for the biopsy urease test: Christensen's urea broth (for the first 600 patients) and the Christensen's urea broth modified by increasing the concentration of phenol red and omitting the nutrients, glucose and peptone (for the remaining patients). Both the Christensen's urea broth and modified urea broth were almost 100% specific when compared with detection of C pylori by Gram, culture and histopathology. The modified broth was more sensitive (96% sensitivity compared with culture) than the Christensen's broth (92% sensitivity) but this difference was not statistically significant. The modified broth gave significantly more positive results (58%) in less than 30 minutes than the Christensen's broth (48%). Seventy four per cent of positive results were available in less than two hours. Specimens from patients with extensive C pylori infection gave more rapid results: 86% of specimens that yielded a profuse growth of C pylori and 76% that contained numerous organisms on histological sections had a positive urease test in less than one hour. There was no significant difference between the specificity and sensitivity of our modified urea broth and the other modified broths described in the literature. This test is a cheap and rapid alternative to the diagnosis of C pylori by Gram stained smears or culture.

New spiral bacterium in gastric mucosa.

A new spiral bacterium, distinct from Campylobacter pylori, was found in the gastric mucosa of six patients with gastrointestinal symptoms. All patients had chronic active type B gastritis and four had oesophagitis. Culture and microscopy for C pylori infection was negative. These unculturable spiral organisms were probably an incidental finding in patients presenting for upper gastrointestinal endoscopy, but it is not possible to say from this small series whether these organisms cause chronic active gastritis. The organism is helical, 3.5-7.5 microns long and 0.9 micron in diameter with truncated ends flattened at the tips, and up to 12 sheathed flagella 28 nm in diameter at each pole. It is proposed that this spiral bacterium should be called "Gastrospirillum hominis Gen.nov., Sp.nov."

Proliferative glomerulonephritis in mice given intravenous Corynebacterium parvum.

Mice given a killed suspension of Corynebacterium parvum (C.p.) developed nephritis as part of an immune complex disease. The nephritis was dose-related. After a single dose of 70 microgram (a human-equivalent dose) or of 466 microgram there was a mesangiopathic glomerulonephritis and after repeated human-equivalent doses there was a mesangiocapillary glomerulonephritis. Antibodies to C.p. increased and circulating immune complexes were detected. Mice receiving repeated doses also developed an arteritis. Study of this model may help in the understanding of human immune complex disease and the pathogenesis of glomerulonephritis.

Immune-complex disease in mice and humans given C. parvum.

The present studies in mice and cancer-bearing patients, treated with C. parvum (CP) immunotherapy, were to determine the effects of CP on the production of immune complexes (IC) and associated disease. Using the Clq-binding assay, circulating immune complexes were detected in mice given a single high dose of CP (466 microgram) and repeated human-equivalent doses (70 microgram). All mice treated with CP developed proliferative glomerulonephritis, the severity of which was dose-related. The histological and immunofluorescent patterns of the nephritis were those attributed to immune-complex disease. The mice had haematuria but were not in renal failure. Fifty patients with inoperable lung cancer were studied. All received radiotherapy. Twenty-two had no other treatment (controls) and 28 were treated with infusions of CP. Using 2 immune-complex assays (Clq binding and monoclonal rheumatoid-factor binding) IC were found in 10/22 control patients but these did not develop haematuria or proteinuria. Twenty-four of the 28 patients treated with CP developed transient haematuria and/or proteinuria with red-cell and hyaline casts, the changes resolving over 5 days. Immune complexes were detected in 5 of these 28 patients before CP treatment. Although 16/28 had IC at the time of haematuria and proteinuria, these findings were difficult to interpret because IC may occur in response to the tumour, the radiotherapy, or the CP. Although no patient developed renal failure, we believe that those treated with CP should have regular assessment of their renal function.

In vitro fixation of guinea pig complement by renal biopsies.

The in vitro fixation of heterologous complement by cryostat sections of human renal biopsy material was studied to determine the mechanism of complement activation. Various types of guinea pig sera with different parts of the complement system inhibited were used, the fixation of complement being detected by direct immunofluorescence. Cases of idiopathic focal nephritis with mesangial IgA (mesangial IgA disease), Henoch-Schönlein purpura (HSP) and mesangio-capillary glomerulonephritis (MCGN) fixed complement by the alternative pathway alone and in systemic lupus erythematosus (SLE) both the classical and alternative pathways were involved. Only one of the seven cases of membranous glomerulonephritis fixed complement and this was by the classical pathway. After prior treatment with C3b inactivator, the in vitro complement fixation in mesangial IgA disease, HSP and MCGN was greatly reduced. In SLE it was slightly reduced and in membranous glomerulonephritis there was no change. This is a convenient method of studying the biological properties of complexes which is believed to reflect the in vivo behaviour of the tissue deposited complex.