Pathology underrates colon cancer extranodal and nodal metastases; ex vivo radioimmunodetection helps staging.

PURPOSE: Colorectal carcinoma is frequently accompanied by small lymph nodes metastases that often escape pathologic examination. We evaluated whether ex vivo radioimmunodetection with the Affinity Enhancement System (AES) could improve detection of mesocolonic metastases. EXPERIMENTAL DESIGN: A bivalent 111In-labeled hapten was injected (16 patients) 4 days after a bispecific antibody (anticarcinoembryonic antigen, antihapten). Surgery was done 1 to 3 days later, and radioactive uptake in the mesocolon was recorded. Extensive pathologic examination of the mesocolon (reference method) was done after fat dissolution. This method visualizes all lymph nodes but is not in routine use. RESULTS: The reference method disclosed 705 nodes. There was no significant difference between the number of node metastases detected by AES or by the reference method (16 versus 17). Better detection would have been obtained by AES than by routine pathology (P<0.01). In addition 12 extranodal metastases were found in this study of which eight were detected by AES. The prognostic importance of such extranodal metastases has been underlined in the literature. Routine pathology combined with AES would have disclosed all node metastases and 86% of total metastases versus 35% by routine pathology alone. CONCLUSIONS: Ex vivo radioimmunodetection could improve nodal and extranodal metastases detection in patients with colorectal cancer. Its value for improving pathologic analysis, together with the effect of these small metastases on prognosis, should be further evaluated. The benefit of adjuvant chemotherapy for patients upstaged with radioimmunodection should also be assessed because adjuvant chemotherapy improves the 5-year survival of stage III patients.

The results of successful penetrating keratoplasty using donor organ-cultured corneal tissue.

BACKGROUND: The aim of this study was identification of predictive factors for postoperative visual acuity in patients with a clear organ-cultured graft and to analyze the change in visual acuity between 12 and 24 months after transplantation. METHODS: The study design was a prospective cohort study. A total of 342 consecutive penetrating keratoplasties using donor organ-cultured grafts, performed in 324 patients, were included. Visual acuity, graft thickness, and graft endothelial cell density were recorded in patients with clear transplants. RESULTS: At 24 months postoperatively, 25 (18.7%) of 134 patients had 20/200 or worse visual acuity and 66 (49.3%) had 20/40 or better visual acuity. Graft thickness took 1 month to decrease to normal values. A temporary graft thinning occurred at 6 months postoperatively, followed by recovery of normal graft thickness by 18 months. The average postoperative endothelial cell density was 1,533+/-598 cells/mm2 during the second year. The 24-month LogMAR (logarithm of minimal angle of resolution) visual acuity correlated with preoperative LogMAR visual acuity (beta=0.26, P=0.005), postoperative lens status (beta=-0.34, P=0.008), preoperative intraocular pressure (beta=0.50, P=0.020), and postoperative astigmatism (beta=0.17, P=0.040). Visual acuity (P=0.022) significantly improved between 12 and 24 months. Preoperative diagnosis (P < 0.0001) and postoperative lens status (P < 0.0001) significantly influenced the change in LogMAR visual acuity between 12 and 24 months. CONCLUSIONS: Donor variables do not influence the visual acuity results of penetrating keratoplasty using organ-cultured donor tissue, whereas they have a strong influence on graft survival and graft endothelial cell density. Visual acuity improves during the first 2 years after transplantation. After keratoplasty, organ-cultured corneal grafts undergo dramatic modifications of their thickness and probably of their transparency.

Cryopreservation and culture of human corneal keratocytes.

PURPOSE: To assess the effects of two different concentrations of albumin in a cryoprotective solution and two freezing methods on human corneal keratocyte ctyopreservation. METHODS: Isolated keratocytes were used for cryopreservation. Solutions of 10% dimethylsulfoxide with either 2% or 10% human albumin were used as cryoprotective agents. Cells either were transferred directly into a -80 degrees C freezer (freezing rate, 2 degrees C/min) or were cooled in a programmed freezer (1 degrees C/min until -40 degrees C and then 10 degrees C/min), which resulted in four different cryopreservation protocols. Cells were stored at -80 degrees C, then were thawed at 37 degrees C, and subsequently were cultured. Keratocytes were studied by means of trypan blue staining, growth assay, apoptosis assays, transmission electron microscopy, and immunochemistry. RESULTS: The percentage of cells that were alive after thawing ranged from 80% to 99% by trypan blue staining and from 45% to 60% by flow cytometry. The ratio of the number of living cells at the end of primary culture after cryopreservation to that before cryopreservation was significantly (P=0.04) higher after direct transfer into the -80 degrees C freezer than after controlled-rate freezing, whereas the albumin concentration had no significant influence on this ratio (P=0.45). The percentage of apoptotic cells was significantly higher after cryopreservation than in the control group of noncryopreserved cells; more than 5% 24 hours after thawing. Cryopreservation did not modify the keratocyte ultrastructure. Fibroblast growth factor dramatically decreased the serum-induced cell expression of alpha smooth muscle actin, whereas cryopreservation had no influence on this cell expression. CONCLUSIONS: A freeze-thaw trauma, which was related to cryopreservation-induced cell apoptosis, was revealed during primary culture after thawing. Direct transfer into the -80 degrees C freezer resulted in better postcryopreservation growth in the culture than controlled-rate freezing. A change in albumin concentration from 2% to 10% did not affect the results.

Prediction of spectacle-corrected visual acuity using videokeratography.

PURPOSE: Our aim was to improve prediction of spectacle-corrected visual acuity (SCVA) using indices derived from the EyeSys System 2000 data (version 3.1). METHODS: We studied corneal topography in 182 eyes from 8 groups of patients. Holladay Diagnostic Summary indices were recorded. Nine statistical indices calculated with the first 8-ring data and refractive power symmetry index were also studied. Correlation with SCVA (LogMAR units) was studied by means of Pearson's regression. Multiple linear regression was used to obtain linear equations combining several indices. RESULTS: At a univariate level, total astigmatism cylinder showed the strongest correlation with SCVA (r = .63, P = .0001). At a multivariate level, the predicted visual acuity obtained by linear equation combining the asphericity coefficient, the predicted corneal acuity, the mean of the means, and the total astigmatism cylinder was closely associated with SCVA (r = .72, P = .0001). It was identical to SCVA in 58.2% of the cases, within one line in 75.8%, and within two lines in 91.2%. CONCLUSION: Multiple linear regression resulted in the best prediction of spectacle-corrected visual acuity, giving notable improvement in prediction of spectacle-corrected visual acuity as compared to the predicted corneal acuity available in the EyeSys System 2000.

The triple procedure: in the bag placement versus ciliary sulcus placement of the intraocular lens.

AIMS: To evaluate the influence of intraocular lens (IOL) placement on triple procedure clinical results and to investigate whether it is appropriate to use phacoemulsification in patients with large lens nucleus. METHODS: 40 consecutive penetrating keratoplasties combined with cataract extraction performed in a single institution were studied. Whenever possible a capsulorhexis was performed and the IOL was placed into the capsular bag. Phacoemulsification was used when the nucleus was too large to pass through the capsulorhexis. RESULTS: Out of 25 patients with an intact capsulorhexis phacoemulsification was used in 13 (52.0%) whereas the entire nucleus passed through the capsulorhexis in the remaining 12 patients (48%). The average 12 month visual acuity was 0.46 (SD 0.21) in patients with in the bag IOL (n = 23) and 0.29 (0.08) in patients with ciliary sulcus IOL (n = 13) (p = 0.04). Elevated intraocular pressure occurred in 26.1% (6/23) of patients with in the bag IOL and 61.5% (8/13) of patients with ciliary sulcus IOL (p = 0.08). The average postoperative graft thickness at 18 months was 552 (27) microns in the former group and 650 (29) microns in the latter group (p = 0.04). No significant difference in graft survival, postoperative endothelial cell density, astigmatism, and videokeratoscopic measurements was found between both groups. CONCLUSION: In the bag placement of the intraocular lens during the triple procedure results in better outcome of transplantation than ciliary sulcus placement of the IOL. Phacoemulsification allows removal of large nuclei through a 5 mm capsulorhexis without performing relaxing incisions out towards the periphery of the capsule.

Donor organ cultured corneal tissue selection before penetrating keratoplasty.

AIMS: Donor organ cultured corneal tissue selection before penetrating keratoplasty is carried out by taking into account different variables. The objective was to identify preoperative variables which are significantly and independently associated with transplant outcome and should effectively be taken into account before transplantation. METHODS: 231 consecutive penetrating keratoplasties were prospectively studied using organ cultured tissue. Morphometric analysis of the donor corneal endothelium was performed before transplantation. Graft survival and endothelial cell density, during the second year following transplantation, were studied both at a univariate and multivariate level. RESULTS: Recipient age, recipient rejection status, and preoperative diagnosis significantly influenced graft survival. Graft survival was higher when using corneal tissue from donors older than 80 years. Postoperative endothelial density decreased with preservation time and coefficient of variation after preservation. It increased with endothelial cell density after preservation and deswelling time, and correlated with preoperative diagnosis. CONCLUSION: Organ cultured corneas with endothelial cell density after preservation < 2000 cells/mm2, and high coefficient of variation, may be discarded before transplantation. Corneas should be preserved for less than 3 weeks, and allowed to deswell before transplantation for 2 or 3 days rather than 1 day.

Screening human donor corneas during organ culture for the presence of guttae.

AIMS: To detect the presence of guttae by means of light microscopy during organ culture and to evaluate the influence of the presence of guttae in the donor tissue on transplantation outcome. METHODS: Donor corneas were investigated for the presence of guttae by means of light microscopy at the end of organ culture. Recipient corneal buttons from patients with severe Fuchs' dystrophy and donor corneas with advanced guttae were first studied by light microscopy and subsequently by transmission electron microscopy. Lastly, 168 consecutive donor corneas were evaluated for the presence of guttae and issued for transplantation. RESULTS: Corneal specimens with Fuchs' dystrophy displayed numerous round highly reflecting guttae at the level of the corneal endothelium. Donor corneas with advanced guttae showed less numerous guttae. Among 168 organ cultured donor corneas issued for transplantation, low density guttae were found in 43 (25.6%) corneas. The endothelial cell density and figure coefficient were significantly lower and organ culture time was significantly higher in the cornea guttata group than in the control group. The presence of grouped guttae significantly decreased the adjusted graft survival. The incidence of postoperative stage 3 cornea guttata was significantly higher when grouped guttae were found (5/6) than when no guttae or scattered guttae were found (8/101). CONCLUSION: Cornea guttata can be detected during organ culture by means of light microscopy. It is associated with a decrease in endothelial cell figure coefficient and cell density. The presence of grouped guttae is associated with poorer graft survival and more frequent stage 3 cornea guttata in the graft after transplantation.

Automated keratoconus detection using the EyeSys videokeratoscope.

PURPOSE: To evaluate the effectiveness of indices derived from the EyeSys System 2000 in detecting keratoconic corneas. SETTING: Department of Ophthalmology, Hôpital Saint Antoine, Paris VI University, Paris, France. METHODS: Topographies of 208 corneas were evaluated. The corneas were from 8 groups of patients classified by the following diagnoses: normal, regular astigmatism, cataract, radial keratotomy, photorefractive keratectomy, myopic keratomileusis, penetrating keratoplasty (PKP), and keratoconus. Nine statistical indices derived from EyeSys data, 2 Holladay Diagnosis Summary indices (coefficient of uniformity and coefficient of asphericity [Asph]), and our refractive power symmetry index were studied. A training set of 104 corneas was used to determine the most efficient threshold value of each index based on sensitivity and specificity curves. Decision trees combining 2 indices were generated. Sensitivity and specificity were calculated in a validation set composed of the remaining 104 corneas. RESULTS: Based on the results of the training set, the optimum indices were SDSD (standard deviation of the standard deviations of the radii of curvature of each ring) and Asph. In the validation set, the decision tree using these indices featured 88.5% sensitivity and 94.9% specificity; the 4 false-positive cases were in corneas in the PKP group of patients. CONCLUSIONS: Clinically apparent keratoconus can be detected among normal corneas and irregular corneal shape patterns using the EyeSys System 2000 data and a decision tree combining 2 indices.

[Cultures of corneal epithelium on hydrogel for synthetic epikeratoplasty]. / Cultures d'épithélium cornéen au contact d'un hydrogel destiné à l'épikératoplastie synthétique.

PURPOSE: To improve the LLH480 hydrogel cytocompatibility and to assess the interactions between corneal epithelium and biomaterials. METHODS: Rabbit corneal epithelium organotypic cultures were carried out on hydrogel samples coated with fibronectin, laminin, type IV collagen, or heparan sulfate proteoglycan. The control group consisted of epithelial cultures carried out on hydrogel with no coating. Cellular migration was quantified, and statistically analyzed. 8-day cultures were processed for transmission electron microscopy. RESULTS: Epithelial migration on hydrogel was significantly increased (p < 0.001) in the heparan sulfate proteoglycan group, significantly decreased (p < 0.05 and p < 0.001) with laminin or type IV collagen coating, and showed no significant modifications in the fibronectin group. Transmission electronic microscopy showed a monolayer of epithelial cells adherant to the surface of the hydrogel. Deposit of extracellular matrix and sketch of adhesion focal points were also noted. CONCLUSION: LLH480 cytocompatibility can be improved by heparan sulfate proteoglycan coating.

[Astigmatism after penetrating keratoplasty. Videokeratoscopic analysis on a series of 60 grafts]. / Astigmatisme après kératoplastie transfixiante. Analyse vidéo-kératoscopique sur une série de 60 greffes.

PURPOSE: To study the factors which induce post keratoplasty astigmatism. To assess the reliability of different methods in the astigmatism measurement and to study the visual acuity predicting factors. METHODS: We retrospectively studied the corneal topography in 60 eyes with penetrating keratoplasty after suture removal, using the CAS* (Eye Sys). The diagnosis was keratoconus in 65% of the cases and bullous keratopathy in 15% of the cases. The graft was secured with a single running suture in 38.3% of the cases, interrupted sutures in 23.3% of the cases, or a combination of both running and interrupted sutures in remaining 28.3%. RESULTS: The suture method, diagnosis and surgeon did not influence subjective refraction nor visual acuity. The topographic pattern correlated with subjective cylinder (rs = 0.52 p = 0.02). The refractive power cylinder ("Holladay diagnostic summary") correlated well with subjective cylinder (rs = 0.81 p < 0.001) and visual acuity (rs = -0.63; p < 0.001). The Javal keratometry remains the best method to measure astigmatism axis (rs = 0.58; p < 0.001). CONCLUSIONS: The Holladay diagnostic summary (refractive power) is a useful tool for evaluating qualitative outcome of corneal transplantation.

Modelling and analysis of 3-D arrangements of particles by point processes with examples of application to biological data obtained by confocal scanning light microscopy.

Within the concept of point processes, a review is presented of quantities which can be used in studies of three-dimensional (3-D) aggregates of particles. Suitable characteristics and estimators are given for both unmarked and marked point processes. To demonstrate the feasibility of such quantitative approaches, an application in histology, dealing with 3-D arrangements of cell nuclei in rat liver, is described. Using a confocal scanning light microscope, 3-D images are recorded and image analysis used to obtain the coordinates of the centroid, together with the volume and DNA content, of each cell nucleus. Examples of results are given, using both unmarked and marked point processes. In the latter case, cell type, nuclear volume and ploidy group are suitable marks.

Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of Bcl-2 protein family.

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.

Automatic cell culture quantitation with TRAKCELL: application to cell toxicology and differentiation.

An automated system, TRAKCELL, was developed for the quantitation of cells in culture. It enabled cell counting, classification according to morphological cell characteristics and measurement of cell proliferation and differentiation. The system was tested on the toxic effect of ascorbic acid on rat brain catecholaminergic neurons in primary culture. In parallel, the effects of nerve growth factor, dexamethasone and forskolin on cell differentiation were studied using rat pheochromocytoma PC12 cells. The results show that the system permits rapid and reproducible measurements of cell density and of the morphological changes observed following various drug treatments.

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks.

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 microns deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-500 (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.