Analysis of the distribution of MRI contrast agents in the livers of small animals by means of complementary microscopies.

BACKGROUND: Magnetic resonance imaging (MRI) contrast agents contain magnetic molecules such as iron (Fe) or gadolinium (Gd) that are injected in vivo into rats or mice to study their distribution inside the liver. Fluorescent europium (Eu) can be used as a model of Gd to obtain comparable information of this distribution of corresponding contrast agents. In a similar approach, Fe can be attached to Texas Red and used as a model of ferumoxides and be detected by fluorescence. METHODS: To combine and compare the advantages of different microscopic imaging modes, characterization studies were carried out by means of a confocal laser scanning microscope (CLSM), a secondary ion mass spectrometric (SIMS) microscope, and an electron energy loss spectrometric (EELS) microscope. In the case of CLSM, the locations of fluorescent signals inside preparations were determined by factor analysis of biomedical image sequences (FAMIS) and selection of image sequences at emission. RESULTS: By CLSM and FAMIS, we distinguished chelated Eu and Texas Red attached to Fe. By SIMS microscopy, we distinguished Eu and Gd of chlorides and chelates and Fe of a ferumoxide. By EELS microscopy, we distinguished Eu and Gd of chlorides. CONCLUSIONS: Analysis of compounds inside correlative specimens by means of CLSM, SIMS, and EELS microscopes provided complementary results.

Conversion from cyclosporin A to tacrolimus in pediatric liver transplantation.

The dosing regimen for conversion from cyclosporin A (CsA) to tacrolimus immunosuppression was studied in 12 pediatric liver allograft recipients. Patients were stratified according to their age. Tacrolimus was started orally at a dosage of 0.05 mg/kg b.i.d., 12 h after stopping CsA administration and increased thereafter if needed. Tacrolimus and CsA concentrations were assayed by immunoassay using, respectively, an IMx and a TDx autoanalyzer (Abbott-France). Mean CsA concentration was in the therapeutic range 12 h prior to and at the time of introduction of tacrolimus. After 72 h of tacrolimus therapy, CsA concentrations were undetectable whereas mean tacrolimus concentration was 10.4 ng/mL with a mean dose of 0.09 mg/ kg b.i.d. The decline of CsA concentration was clearly biphasic. A slower decline in CsA concentration was detected after initiation of tacrolimus therapy, suggesting an inhibition of CsA metabolism by tacrolimus. No nephrotoxicity was observed. This dosage regimen allowed effective immunosuppression while avoiding additive nephrotoxicity.

[Large cell variant of small cell carcinoma of the ovary with hypercalcemia]. / Variante à grandes cellules d'un carcinome à petites cellules de l'ovaire avec hypercalcémie.

We describe an original distinct type of ovarian small cell carcinoma: large cell variant. The distinctive histologic features of tumor cells were the presence of large nuclei with prominent nucleoli and abundant eosinophilic cytoplasm. Immunohistochemistry revealed strong diffuse vimentin, smooth muscle actin positivity and slight reactivity with epithelial markers. Electron microscopy showed aggregates of intermediate filaments, intercellular attachments and no dense core granules. This tumor is associated with paraendocrine hypercalcemia in two thirds of cases. Parathyroid hormone-related protein was focally positive. This tumor is characterized as a very lethal neoplasm, occurring primarily in young women.

SIMS microscopy: methodology, problems and perspectives in mapping drugs and nuclear medicine compounds.

Secondary ion mass spectrometry (SIMS) microscopy, a mass spectrometry method designed in the 1960s, offers new analytical capabilities, high sensitivity (ppm to ppb region), high specificity and improved lateral resolution, thus facilitating insight into many physiological and biomedical questions. Apart from the sample preparation and the physical characteristics of the detection, the biological model must also be considered. SIMS analysis of diffusible ions and molecules requires strict cryogenic procedures which always begin by a flash-freeze fixation. Cellular integrity can be checked by mapping the major element distributions since intra and extracellular ions are redistributed only in damaged cells. Cryofixing may be followed either by a freeze-fracture methodology or by cryoembedding and dry-cutting. Chemical sample preparation is only used for ions or molecules bound to fixed cell structures. The use of scanning procedures ameliorates the lateral resolution and chromosome imaging has been reported with probe size of below 50 nm. Absolute quantification can be derived for embedded specimen by using internal references included in tissue equivalent resins. The sensitivity is limited by the ionization yield of the tag element and may be further impaired when working at high mass resolution (> or = 5000) to eliminate interfering cluster ions. SIMS drug mapping is usually performed after in vitro administration of a molecule to cell culture systems. Drug detection is accomplished indirectly by detecting a tag isotope naturally present or introduced by labelling, mainly with halogens, 15N and 14C. Molecular imaging with TOF-SIMS is an appealing alternative especially for heavier compounds. We stress some biological problems through a critical review of published SIMS drug studies. SIMS proved useful in assessing the targeting specificity of nuclear medicine pharmaceutics, even after in vivo administration. The first microscopic evidence of a thionamide induced follicular blockade of the iodine organification process is presented in a human sample.

Mutual contribution by "track" microradioautography method (MRA) and secondary ion mass spectrometry (SIMS) microscopy: prospects in technetium dosimetry at-the cellular level.

The determination of cellular uptake sites of radioligands used for cell labelling for diagnostic purposes is an essential prerequisite for evaluating the radiation dose to the cell nucleus and cytoplasm. The distribution of 99mTc-HMPAO in labelled leukocytes was studied by two microscopic imaging techniques on the same biological material: the "track" microradioautographic method (MRA) and Secondary Ion Mass Spectrometry (SIMS) microscopy. The "track" method used internal conversion electrons of 99mTc, leading to the formation of silver grains in a thick layer nuclear emulsion deposited onto cellular smear. In order to improve the specificity of the "track" detection, a minimum of 5 consecutive silver grains was required. In SIMS Microscopy, mapping with 99Tc "daughter" nuclide of 99mTc (half-life: 2.13.10(5) years) was obtained after sputtering of superficial molecular layers on embedded specimen sections. A mass resolution of about 5,000 was needed to circumvent polyatomic ion interferences. Both methods were able to demonstrate a very heterogeneous distribution of technetium from one cell to another. The sensitivity and signal/noise ratios were excellent for both methods. The lateral resolution of SIMS microscopy (0.5 microns) was far better than that of MRA. Therefore, only SIMS is able to distinguish between nuclear and cytoplasmic localization. On the other hand, quantification was not achieved for SIMS, although semi-quantification is possible with MRA. The field of view of MRA is far larger, allowing a better statistical approach for quantification. Both methods appear to be complementary to determine the distribution of technetium at the cell level. MRA is simpler and better fitted to the study of a cell population or a tissue. The unique spatial resolution of SIMS allows to focus the study on subcellular structures.

Bile levels of carcino-embryonic antigen in patients with hepatopancreatobiliary disease.

OBJECTIVE: To evaluate the value of biliary carcino-embryonic antigen (CEA) in the differential diagnosis of malignant and benign hepatopancreatobiliary disease. PATIENTS: One hundred patients were prospectively studied. Benign diseases were present in 39% of the patients while 61% had malignant diseases. METHODS: Samples of serum were taken from all patients just before endoscopic retrograde cholangiopancreatography (ERCP) and samples of biliary CEA were obtained during ERCP. RESULTS: The sensitivity of serum CEA and carbohydrate antigen 19-9 (CA 19-9) in detecting malignancy were 50% and 92%, respectively, while the respective specificities were 95% and 72%. The mean biliary CEA level of the benign group was significantly different from that of the malignant group (35.7 +/- 8.7 ng/ml vs 268 +/- 85.5 ng/ml), but there was considerable overlap between the two groups. With a cut-off level of 20 ng/ml, the sensitivity and specificity were 84% and 64% respectively. The mean bilirubinaemia value was significantly higher in malignant disease than in benign disease (57.4 +/- 13.9 mumol/l vs 235 +/- 19.8 mumol/l). Multidimensional analysis indicated that only bilirubinaemia (P < 109-3)) was independently predictive of malignant disease. CONCLUSION: Biliary CEA assessment seems useless in distinguished between benign and malignant causes of cholestasis.

Antibodies against p53 protein in serum of patients with benign or malignant pancreatic and biliary diseases.

Specific markers for pancreatic or biliary cancer have been developed in the past few years. Ca 19-9 has a good sensitivity but it is also increased in benign cholestasis. Mutations in the p53 gene are commonly reported in pancreatic cancer and can be detected by a serological analysis. The aim of this work was to find out the sensitivity and specificity of this new assay in diagnosing cancer of the pancreas or of the bile ducts. The presence of antibodies against p53 was determined by an enzyme linked immunosorbent assay (ELISA) in 29 patients with pancreatic cancer, 33 with biliary tract cancer, and 33 with benign biliary or pancreatic diseases as controls. p53 Antibodies were detected in eight of 29 patients with pancreatic cancer (28%), in five of 33 patients with biliary tract (15%), and in one patient (3%) with stones of the common bile duct. The sensitivity and the specificity for the diagnosis of malignant biliary or pancreatic diseases were 21% and 96% respectively. It is concluded that the presence of p53 antibodies in the serum of patients with pancreatic and biliary diseases is specific for malignancy and independent from the presence of cholestatic disease.

Recent developments in medical applications of SIMS microscopy.

The purpose of this review is to present the recent developments in the medical applications of SIMS microscopy. This technique is one of the microanalytical mass spectrometry methods which allow in theory the detection of all the elements of the Mendeleiev table as well as the separation of stable and radioactive isotopes. It is based on a phenomenon whereby a biological sample surface is sputtered by bombardment with an energetic 'primary ion' beam. Part of the sputtered matter is ionized and the resulting 'secondary ions' are characteristic of the atomic composition of the analyzed area. These secondary positive or negative ions are collected and separated in a mass spectrometer at low or high mass resolution, which is dependent on both the element studied and its concentration. An analytic image which conserves the tissue distribution of the selected element is displayed on a fluorescent screen linked to an image processing system. Local elemental concentration can also be measured. Results are highly dependent on the techniques used for sample preparation which should preserve both the chemical and the structural integrity of the tissue. Further, the ionic images must be correlated with corresponding images of the same areas of the serial sections observed in a photonic microscope. With our SIMS microscope (lateral resolution approximately 0.5 microns, and mass resolution 300 to 12,000) we have demonstrated that this microscopic imaging technique is suitable for physiopathological studies. We revisited thyroid iodine metabolism by mapping chemical elements such as 32S and 127I, characteristic of hormonal physiology. Newly organified iodine (radioiodine) can be evaluated in relation to previously stored iodine (127I) in a given follicle, thus allowing an appraisal of glandular adaptation to aging and iodine overload. Another area in which SIMS can be used in medicine, is for the localization of drug markers in tumor tissue (e.g. fluorine-5-fluouracil, iodine in iododeoxyrubicin). This could facilitate the evaluation of the intratumor drug concentration at the onset of the treatment. Likewise, SIMS can be used to localize radiopharmaceuticals used in diagnosis (e.g. technetium) and therapy (131I of metaiodobenzylguanidine). This would permit a better evaluation of the radiation dose delivered to tissue. Further prospects are within reach with the imminent advent of higher lateral resolution (0.05 microns) SIMS microscopes.

[Radio-isotope assays of vitamin B 12: value of the Kappa test in a multicenter evaluation]. / Dosages radio-isotopiques de la vitamine B12: intérêt du test Kappa dans une évaluation multicentrique.

Owing to the lack of a reference technique and of an international cobalamin (vitamin B12) standard, and the large discrepancy between laboratory norms, the authors performed a multicentric study to compare five RIA kits usually used. First, classical tests were used to evaluate the analytical performances of each kit. Results did not demonstrate any superiority of one kit over another. Secondly, B12 values were classified among three categories (low, normal and high) characterized by laboratory and then manufacturer norms. The concordance between these two "judgments" was evaluated with the Kappa coefficient. In addition, the Kappa index proved that the norms supplied by the manufacturer were better than those of laboratories. But mean Kappa coefficient established for each norm gave us an unsatisfactory result. Third, clinical informations allowed to improve the classification of the patients. New limits were defined for each technique and should be tested further, routinely in each laboratory.

SIMS microscopy imaging of the intratumor biodistribution of metaiodobenzylguanidine in the human SK-N-SH neuroblastoma cell line xenografted into nude mice.

Microdosimetric evaluations of targeted radiotherapy of neuroblastoma with metaiodobenzylguanidine (MIBG) require precise assessment of the intracellular and intratumor distribution of the drug. We report the use of secondary ion mass spectrometry (SIMS) microscopy, a technique capable of mapping any chemical element within a biological specimen, to determine 127I-MIBG biodistribution in human neuroblastoma SK-N-SH xenografted into nude mice. Highly specific images of 127I-MIBG biodistribution were mapped within the tumor after in vivo administration of the drug and sample processing with cryotechniques (high-speed freezing and cryo-embedding), which prevent MIBG diffusion from original sites of uptake. We showed that the biodistribution of the tracer was highly nonuniform within the tumor. At the cellular level, most of the drug accumulated in the cytosol and perinuclear areas. In contrast, chemical sample processing provided not only a considerable loss in sensitivity due to passive diffusion of the drug in the organic solvents, but also artefactual images mainly due to MIBG redistribution onto the cell nuclei. Based on our findings in this SK-N-SH experimental tumor model, we suggest that MIBG should be attached to long-range emitters, in the hope of irradiating the many tumorous areas that remain carrier-free.

Significance of secondary ion mass spectrometry microscopy for technetium-99m mapping in leukocytes.

Secondary ion mass spectrometry (SIMS) microscopy is the only method potentially capable of mapping all the elements in the periodic table including stable and radioactive isotopes. We used this method to study 99mTc distribution by detecting and localizing of 99Tc, a daughter product which has the same mass and the same chemical properties. It was combined with albumin macroaggregates or with Hexamethylpropylene-amine oxime (HMPAO) in leukocytes. The efficiency of 99Tc ionization under Cs+ bombardment was higher than with an O2+ beam. By using high mass resolution we succeeded in detecting and localizing 99Tc in cell sections by eliminating polyatomic ions that arise from this biological matrix. The 99Tc specific signal was obtained with a mass resolution of 2000 for labeled albumin macroaggregates, and 5000 for HMPAO-labeled leukocytes. In the latter, the labeling varied from one cell to another and 99Tc was present in both the nucleus and the cytoplasm. The results indicate that SIMS microscopy can provide new insights into 99mTc dosimetry.

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.

[Lymphangioma of the orbit]. / Les lymphangiomes de l'orbite.

Three cases of orbito-palpebral lymphangioma are reported. The three cases all have the common features: onset in early childhood, slow progression and extension of the tumor to other structures (palate and temporal fossa). The usual complications (hemorrhage and cellulitis) were documented in our cases. Biopsy of the lesions showed variably sized endothelial lined spaces without a capsule. Theses spaces contain eosinophilic material without blood elements. In two cases, chirurgical excision gave orbital edema. The authors think that orbital lymphangioma is a clinical entity. The treatment is difficult and small repeated excisions and cryotherapy are recommended.