A new approach for the analysis of testicular cells using a laser scanning cytometer.

The laser scanning cytometer (LSC) is a new laboratory tool that offers increased sensitivity and specificity compared to traditional technology. By combining the properties of the advantages of flow cytometry and immunohistochemistry, LSC-based analysis allows the automated evaluation of testicular cells in general and meiosis in particular. Testicular cell smears with previous staining by propidium iodide were analyzed by LSC. The results were compared with those for flow cytometry. LSC is a new, applicable methodology for analyzing spermatogenesis schedule.     

Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG-7)

We have demonstrated a method for the in situ determination of the cell cycle phases of TIG-7 fibroblasts using a laser scanning cytometer (LSC) which has not only a function equivalent to flow cytometry (FCM) but also has a capability unique in itself. LSC allows a more detailed analysis of the cell cycle in cells stained with propidium iodide (PI) than FCM. With LSC it is possible to discriminate between mitotic cells and G₂ cells, between post-mitotic cells and G₁ cells, and between quiescent cells and cycling cells in a PI fluorescence peak (chromatin condensation) vs. fluorescence value (DNA content) cytogram for cells stained with PI. These were amply confirmed by experiments using colcemid and adriamycin. We were able to identify at least six cell subpopulations for PI stained cells using LSC; namely G₁, S, G₂, M, postmitotic and quiescent cell populations. LSC analysis facilitates the monitoring of effects of drugs on the cell cycle.     

DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry

OBJECTIVE: To evaluate a rapid and simple method for DNA content analysis of urinary tract epithelial tumors with laser scanning cytometry (LSC). STUDY DESIGN: The subjects were 25 patients (37 specimens) who underwent surgery for urinary tract epithelial tumors. Tissue specimens of such tumors were frozen immediately after tumor resection and stored at -80 degrees C until used. Touch preparations were made and fixed in ethanol at room temperature. The cell nucleus was stained with propidium iodide solution containing RNase, and DNA ploidy was analyzed by LSC. Nuclear debris and overlapping nuclei were gated out by special statistical filters. In LSC, a normal diploid reference peak was determined by observing lymphocytes morphologically on the computer display of the instrument and/or under the microscope. RESULTS: DNA ploidy could be evaluated in all tumor tissues. The time it took from preparing the tumor specimen to the last measurement was about 40 minutes at the shortest, and measurement of all the specimens was completed within one hour. The coefficient of variation was 2.8-7.8% (mean, 4.4%). All eight specimens (100%) at grade 1 (G1) were DNA diploid, but 20% and 85.7% of the G2 and G3 cells, respectively, were DNA aneuploid. In total, 15 of the 37 specimens were DNA aneuploid. All 17 pTa-pT1 specimens (100%) were DNA diploid, but 100% and 50% of the T2 and T3 tumors, respectively, were DNA aneuploid. CONCLUSION: One can now supplement a morphologic diagnosis with useful information using LSC of touch preparations of tumors obtained at surgery or of imprints of archived, frozen specimens. LSC provides excellent DNA histograms for surgical specimens and has great potential for clinical application in pathology.     

Prediction of upper aerodigestive tract cancer by slide-based cytometry.

AIM: To evaluate slide-based cytometry in screening for and following up of carcinoma of the upper aerodigestive tract using swabs for a minimal-invasive approach. METHODS: Laser scanning cytometry (LSC) was used for multiparametric analysis of cells stained for cytokeratin and DNA to determine the DNA-index (DI) of the tumor cells. Histograms with 0.95 < DI < 1.05 and 1.9 < DI < 2.1 were defined as DNA euploid and any other DI as DNA aneuploid. After subsequent HE-staining, single cells were relocalized in order to document morphology. Conventional cytology was also performed on a subset of the slides. Routine histopathology of parallel biopsies served as gold standard in all cases. RESULTS: 115 swabs from 109 patients were obtained from the entire upper aerodigestive tract. 16 swabs were classified as insufficient for LSC. In the remaining 99 specimens, 1 benign lesion was misclassified as malignant, while 61 of the 75 malignant lesions were correctly identified. This corresponds to predictive values of 98.4% and 62.2% for the detection of malignant and benign samples by LSC. CONCLUSION: This pilot study demonstrates the validity of LSC screening for the identification of tumor malignancy in the upper aerodigestive tract from swab collected cytological material.     

Development of a stereological method to measure levels of fluoropyrimidine metabolizing enzymes in tumor sections using laser scanning cytometry.

BACKGROUND: The enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) influence the activities of fluoropyrimidine anticancer drugs. The sensitivity of cancer cells to capecitabine, which is an oral, tumor-selective pre-prodrug of 5-fluorouracil may correlate better to the TP/DPD ratio than to levels of either enzyme alone. Our goal was to develop a quantitative immunofluorescent method for estimating the levels of TP, DPD, and their ratio in archival tumor sections. METHODS: Mouse anti-TP and rat anti-DPD monoclonal antibodies were used for parallel indirect immunofluorescent staining. The fluorescence was measured using a laser scanning cytometer (LSC; CompuCyte, Cambridge, MA) in single cells and in sections prepared from cell lines and a human tumor. The phantom contouring feature of the LSC provided a stereologic approach for collecting the fluorescence intensity data from sections. RESULTS: The relative fluorescence intensities measured in single cells or in sections of the cell lines, using single or double labeling, were similar, supporting the suitability of phantom contouring and two-color staining. Sections of the T-24 and ZR-75-1 cell lines placed on the same slide as the tumor section were used as internal standards for fluorescence measurements. The TP/DPD ratios measured in three cell lines correlated well with the cytotoxicity of 5'-deoxy-5-fluorouridine measured in vitro, indicating that the measurements are related to the biological activity of the drug. CONCLUSIONS: Plotting the data as contour maps of the topologic distribution of fluorescence intensities in tumor sections allows subsequent histopathologic examination, which may reveal features of the tumors leading to high or low ratios of these enzymes. In addition, this method can be used for any drug target/metabolic system where the key components are known and suitable antibodies are available.     

Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry.

BACKGROUND: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. AIM: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. METHODS: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn ADP Flow Cytometer. RESULTS: The pHi clamping conditions were optimized as 140 mM potassium and 10 microM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. CONCLUSION: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.     

Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques were compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short-term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count. In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.     

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.     

Micronuclei assay by laser scanning cytometry

BACKGROUND: The micronuclei (MN) assay is used to assess the chromosomal/mitotic spindle damage induced by ionizing radiation or mutagenic agents in vivo or in vitro. Because visual scoring of MN is cumbersome semi-automatic procedures that relay either on flow cytometry or image analysis were developed: both offer some advantages but also have shortcomings. METHODS: In the present study laser scanning cytometer (LSC), the instrument that combines analytical capabilities of flow and image cytometry, has been adapted for quantitative analysis of MN. The micronucleation of human breast carcinoma MCF-7 and leukemic HL-60 and U-937 cells was induced by in vitro treatment with mitomycin C. Cellular DNA was stained with propidium iodide (PI), protein was counterstained with fluorescein isothiocyanate (FITC). Two approaches were used to detect MN: (a) the threshold contour was set based on the data from the photosensor measuring red fluorescence of PI and MN were identified on the bivariate PI versus PI/FITC fluorescence distributions by their characteristic position; (b) the threshold contour was set on the data from the sensor measuring FITC fluorescence which made it possible, using the LSC software dedicated for FISH analysis, to assay both the frequency and DNA content of individual MN within each measured cell. RESULTS: The capability of LSC to relocate MN for visual examination was useful to confirm their identification. Visual identification of MN combined with their multiparameter characterization that took into an account their DNA content and protein/DNA ratio made it possible establish the gating parameters that excluded objects that were not MN; 93.3+/-3.3 events within the selected gate were MN. It was also possible to successfully apply FISH software to characterize individual cells with respect to quantity of MN residing in them. The percentage of MN assayed by LSC correlated well with that estimated visually by microscopy, both for MCF-7 (r = 0.93) and HL-60 cells (r = 0.87). CONCLUSIONS: LSC can be used to obtain unbiased estimate of MN frequencies. Unlike flow cytometry, it also allows one to characterize individual cells with respect to frequency and DNA content of MN residing in these cells. These analytical capabilities of LSC may be helpful not only to score MN but also to study mechanisms by which clastogenic agents induce MN.     

DNA microspectrophotometry of bone sarcomas in tissue sections as compared to imprint and flow DNA analysis

The aim of the present study was to establish an upper limit of diploidy for microspectrophotometric (MSP) DNA measurements in sections of mesenchymal tissue analyzing DNA data of a large number of normal cell populations. The reliability of this upper limit of diploidy for discriminating between diploid and hyperploid bone sarcomas was tested by analyzing the same tumors by MSP in imprint preparations and flow cytometry (FCM). The median DNA value of control cells in tissue sections was given arbitrary value of DNA index (DI) 1.0, denoting the diploid DNA content. The proportion of cells with DNA values exceeding DI 1.25 (greater than DI 1.25) was determined for each normal cell population. The maximum percentage of cells with DNA values exceeding DI 1.25, encountered by analysis of 91 normal cell populations in tissue sections, was 31%. This percentage was set as an upper limit of diploidy. Hence, tumors with a higher percentage of cells greater than DI 1.25 were classified as hyperploid. When we applied this criterion, 31 of 36 sarcomas analyzed by MSP in tissue sections were hyperploid, which was in complete agreement with FCM and MSP in imprints of the same tumors. Apart from discriminating between diploid and hyperploid tumors, an attempt was made to determine peak DNA values of sarcomas analyzed in tissue sections. Peak DNA values, as defined by a minimum of 30% of the cells within a class width of DI 0.25, could be determined for 23 of 36 tumors. These peak DNA values correlated well with corresponding peaks obtained by FCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of grade of urothelial cell carcinomas and DNA histogram features assessed by flow cytometry and automated image cytometry.

OBJECTIVE: To analyse how DNA ploidy and S-phase fraction (SPF) by flow cytometry (FCM) and an optimised fully automatic DNA image cytometer (ICM) correlate with grade in TaT1 urothelial cell carcinomas (UC) of the urinary bladder. MATERIALS AND METHODS: Two-hundred-and twenty-eight consensus cases were analysed. Single cell suspensions were stained (DAPI for FCM, Feulgen for ICM). There was enough material for both FCM and ICM in 202 of these cases. FCM and optimised ICM measurements were performed on the 202 UCs. To discriminate between different grades, single- and multivariate analyses was performed on DNA histogram features obtained with the MultiCycle program (using DNA index (DI) and SPF). RESULTS: Overall measurement time of the adapted ICM method was 10.7 minutes per case (range 5.9-29.8 min.) and required little additional interactive object rejection (average 152 objects (84-298) on 3000 objects per case measured, which took 9.9 minutes on average, range 8.3-15.5 minutes). The ICM histograms looked much "cleaner" with less noise than the FCM graphs. The coefficient of variation (CV) of the diploid peak for ICM (5.4%) was significantly lower than for FCM (5.9%) (p<0.0001). ICM features were more strongly correlated to grade than FCM features. In multivariate analysis, the best discriminating set of features was DNA ploidy and SPF (both by ICM). CONCLUSIONS: The adapted fully automated DNA ICM works very well for UCs. Low CV DNA ICM histograms are obtained in a time comparable to FCM. The DNA ICM results have stronger discriminative power than DNA FCM for grade in TaT1 UCs.     

“Decoding the Dots”: The ImageStream system (ISS) as a novel and powerful tool for flow cytometric analysis

The aim of this article is to provide a brief review of the ImageStream system (ISS). The ISS technology was developed as a novel method for multiparameter cell analysis and subsequently as a supportive tool for flow cytometry (FC). ISS integrates the features of FC and fluorescent microscopy collecting images of acquired cells for offline digital image analysis. The article presents an overview of the main characteristics of ISS and a comparison between ISS, FC and the laser scanning cytometer (LSC). We reviewed ISS applications focusing on those involved in cellular phenotyping and provide our own experience with using ISS as a supportive tool to classical FC and demonstrate the compatibility between FC and ISS photometric analysis as well as the advantages of using ISS to confirm FC results.     

Laser scanning cytometry can complement the flow cytometric DNA analysis in paraffin-embedded cancer samples: a paradigmatic case

Archival studies on paraffin-embedded tumor samples are often complicated by difficulty obtaining a reliable diploid DNA standard. Nontumor cells, e.g., inflammatory and stromal cells, most often found interspersed among tumor cells, would represent a solution to this problem. Unfortunately, there is an inherent difficulty to positively identifying tumor cells in paraffin-embedded specimens. Using an aneuploid paraffin-embedded breast cancer sample, we show here that laser scanning cytometer (LSC) in conjunction with flow cytometry can help to address this issue. Following standard protocols, the tissue was deparaffinized and rehydrated, and the nuclei mechanically isolated before being exposed to propidium iodide. An aliquot served for single-parameter flow cytometric analysis, and the remaining cells were cytocentrifuged onto a microscope slide and LSC analysis was performed. The DNA histogram profiles generated by the two approaches were comparable and both showed the presence of cell populations with different DNA content. To assess the nature of these subsets, we performed a correlated measurement of DNA content and chromatin organization at the single-cell level by LSC. This allowed the identification of several subsets of nuclei. Slides were then stained with Giemsa and the nature of these subsets was assessed morphologically by exploiting the relocating capability of LSC. Inflammatory and stromal cells, residual diploid epithelial cells, and hyperdiploid tumor cells-each characterized by a peculiar coordinate pattern of DNA content and chromatin organization-could be positively identified. Diploid, nontumor cells can then be used as an internal standard for DNA ploidy.     

DNA ploidy in gastrointestinal B-cell lymphomas. An image analysis study of 43 cases

OBJECTIVE: To analyze the prognostic importance of DNA ploidy pattern on gastrointestinal (GI) B-cell lymphoma using image cytometry (ICM) and to compare the results with previously published flow cytometry (FCM) data. STUDY DESIGN: Forty-three cases of surgically resected primary GI B-cell lymphomas were examined. Thirty-eight tumors were located in the stomach, 2 in the small intestine, 1 in the large bowel and 2 in both the stomach and small intestine. Six cases were at stage E I 1, 15 at stage E I 2, 20 at stage E II 1 and 1 each at stages III and IV. Histologically, the lymphomas were classified as GI low grade marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type (low grade, 12 cases), low grade MALT lymphoma with a high grade component (mixed type, 10 cases) and GI diffuse large B-cell lymphoma (DLBC) (high grade MALT lymphoma, 21 cases). After gross removal of nonneoplastic tissue, single cell suspensions were prepared from paraffin blocks and stained according to Feulgen. Ploidy analysis was done using a custom-made DNA cytometer and Optimas image analysis software (Optimas Corp., Seattle, Washington, U.S.A.). RESULTS: Aneuploidy was found in 42% (5/12 cases) of low grade MALT lymphoma, 90% (9/10 cases) of mixed type lymphoma and 100% (21/21 cases) of GI DLBCL. DNA ploidy had no significant impact on overall survival time (P = .73). CONCLUSION: ICM analysis showed a higher proportion of aneuploidy in GI lymphomas as compared to that in prior studies using FCM for ploidy determination. Whether DNA ploidy is an independent prognostic factor remains to be determined.     

肺鳞癌患者与健康人血清的差异蛋白质组学研究

为筛选肺鳞癌的血清标志物,采用二维凝胶电泳(2-DE)技术分离I期肺鳞癌患者和健康人的血清蛋白质,PDquest图像分析软件识别差异蛋白质点,电喷雾串联质谱(ESI-Q-TOF MS/MS)鉴定差异蛋白,然后应用蛋白质印迹和免疫组化方法分别检测差异蛋白——结合珠蛋白-2(haptoglobin-2,HP-2)在肺鳞癌患者血清和健康人血清以及肺鳞癌组织和癌旁正常支气管上皮组织中的表达.建立了肺鳞癌患者和健康人血清的2-DE图谱,图像分析软件识别了1O个差异蛋白质点,质谱鉴定了4种差异蛋白;蛋白质印迹分析显示,HP-2在肺鳞癌血清中的表达水平显著高于健康人(P<0.05),但其表达水平与肺鳞癌的临床分期无明显相关性;免疫组化结果显示,HP-2在肺鳞癌组织中的表达水平高于癌旁正常支气管上皮组织(P<0.05).研究结果提示:HP-2是候选的肺鳞癌血清分子标志物,血清中HP-2水平对肺鳞癌诊断可能具有一定的参考价值;肺鳞癌组织中HP-2表达上调可能是患者血清中HP-2表达升高的原因之一.     

Application of laser scanning cytometry to the analysis of chromosomal aberrations induced by benzo[a]pyrene in CHO-WBLT cells

BACKGROUND: A recently developed laser scanning cytometry technique was applied to cytometric studies to detect rapidly stable chromosomal aberrations induced by a carcinogen in a Chinese hamster fibroblast cell line, CHO-WBLT. METHODS: Individual chromosomes were collected from metaphase cells by a syringe technique and spread on slides. The DNA content of each chromosome stained with propidium iodide was measured with a laser scanning cytometer (LSC). A characteristic DNA histogram, designated as the "laser scanning karyotype (LSK)," was obtained from about 20,000 chromosomes of CHO-WBLT cells. Each chromosome was confirmed morphologically under the microscope by using a "re-location" system built into the LSC. RESULTS: A total of 21 chromosomes, including marker chromosomes specific to the cell line, were assigned to 10 major peaks in the LSK, which was analogous to the karyotype demonstrated with the classical Q-banding technique. In contrast, clonal sublines isolated after exposure to the carcinogen benzo[a]pyrene showed LSKs different from those found in untreated control cells, and seven of 20 clones were found to be abnormal, with a small number of chromosomal translocations and/or deletions, which were confirmed by Q-banding. CONCLUSIONS: The laser scanning cytometry technique was employed to detect stable chromosomal aberrations in CHO-WBLT cells after treatment with benzo[a]pyrene. The results obtained with this technique were comparable to those obtained by Q-banding; therefore, this method may be useful for rapid primary screening to detect stable, abnormal karyotypes induced by environmental chemicals and/or radiation.     

Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry

BACKGROUND: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria. METHODS: Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC. RESULTS: Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells. CONCLUSION: Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria.     

Grade classification of neuroepithelial tumors using high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy and pattern recognition

Clinical data have shown that survival rates vary considerably among brain tumor patients, according to the type and grade of the tumor. Metabolite profiles of intact tumor tissues measured with high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy (HRMAS (1)H NMRS) can provide important information on tumor biology and metabolism. These metabolic fingerprints can then be used for tumor classification and grading, with great potential value for tumor diagnosis. We studied the metabolic characteristics of 30 neuroepithelial tumor biopsies, including two astrocytomas (grade I), 12 astrocytomas (grade II), eight anaplastic astrocytomas (grade III), three glioblastomas (grade IV) and five medulloblastomas (grade IV) from 30 patients using HRMAS (1)H NMRS. The results were correlated with pathological features using multivariate data analysis, including principal component analysis (PCA). There were significant differences in the levels of N-acetyl-aspartate (NAA), creatine, myo-inositol, glycine and lactate between tumors of different grades (P<0.05). There were also significant differences in the ratios of NAA/creatine, lactate/creatine, myo-inositol/creatine, glycine/creatine, scyllo-inositol/creatine and alanine/creatine (P<0.05). A soft independent modeling of class analogy model produced a predictive accuracy of 87% for high-grade (grade III-IV) brain tumors with a sensitivity of 87% and a specificity of 93%. HRMAS (1)H NMR spectroscopy in conjunction with pattern recognition thus provides a potentially useful tool for the rapid and accurate classification of human brain tumor grades.