[The different types of therapeutic plasma are equivalent?]. / Les différents types de plasmas thérapeutiques sont-ils équivalents ?

In France, three varieties of therapeutic plasma are being processed, distributed and delivered, currently; however, many more varieties are in use worldwide, which go by the property of labile blood component or plasma derived medicines. For one type of component (one given name), several devices and bags and so on are used to concur to its process, which makes that one type of therapeutic plasma may significantly differ from one production setting to one other. This may affect (more or less) the component properties as well as the possibly reported adverse events. This review aims thus, firstly at stressing on the difficulty in comparing data obtained in different contexts, and secondly at making the point on future directions to process therapeutic plasma.

Poloxamines for deswelling of organ-cultured corneas.

BACKGROUND: Poloxamines are amphiphilic tetrofunctional block copolymers composed of four polyoxyethylene-polyoxypropylene arms joined to a central ethylene diamine bridge. Their safe profile allows diverse pharmaceutical and biomedical applications. AIM: To assess their use for corneal deswelling using a porcine model of organ culture (OC). METHODS: Five poloxamines (T90R4, T904, T908, T1107 and T1307) were dissolved in a standard commercial OC medium (control) to reach 350 mosm kg(-1). In vitro cytotoxicity was tested using MTT assay on human corneal epithelial and endothelial cell (EC) lines and on primary human corneal fibroblasts. Paired porcine corneas stored in OC for 3 days were assigned for 48 h to a poloxamine medium or to a standard deswelling medium containing 5% dextran T500. Corneal EC density, morphometry, mortality, stromal thickness and transparency were evaluated before and after deswelling. Post-deswelling, EC viability/mortality was determined using a fluorescent live/dead assay. RESULTS: Besides similar corneal thickness reduction and transparency improvement, T908, T1107 and T1307 decreased EC loss (5.4 ± 1.7% vs. 9.9 ± 2.6% in controls (p < 0.001)) and mortality, improved EC morphometry and reduced endothelial lesions compared to dextran. CONCLUSION: On this porcine model, poloxamines T908, T1107 and T1307 appear as good candidates to replace dextran for the deswelling. Experiments on human corneas are now necessary to confirm their efficiency and safety profile in OC.

[Pathogen inactivation of platelets: organization consequences for platelet transfusion]. / Inactivation/réduction des pathogènes des concentrés de plaquettes: conséquences sur l'organisation de la transfusion plaquettaire.

In the past few years, pathogen reduction technologies for labile blood products have been part of the enhancement of global transfusion safety regarding residual risks of transmitting infectious pathogens. Having carried out a feasibility study for the implementation of pathogen inactivation of platelet concentrates by means of the amotosalen/HCl/UVA (Intercept™) technology, and participated to a reinforced haemovigilance study, we took the opportunity to analyze the organization consequences for platelet concentrates inventory and distribution. This impact study first indicated that those novel needs forced the blood donation service, as well as the labile blood product preparation laboratory, to review and improve practices; secondly, it showed that the routine implementation has little (no major) consequence in the overall organization, independently of the economic consequences (not covered here).

In vitro assessment of apheresis and pooled buffy coat platelet components suspended in plasma and SSP+ photochemically treated with amotosalen and UVA for pathogen inactivation (INTERCEPT Blood System™).

BACKGROUND AND OBJECTIVES: INTERCEPT Blood System™ is a pathogen inactivation system for blood components. The initial approval required a platelet component to be suspended in a combination of plasma and Platelet additive Solution/PAS-III. Improved platelet storage has been reported with Mg++ and K+ supplementation (PAS-IIIM). This study validated the use of INTERCEPT™/PAS-IIIM for apheresis and pooled buffy-coat platelet components. MATERIALS AND METHODS: The platelet dose and pH throughout 5 days of storage met the European and French requirements for quality standards. RESULTS AND CONCLUSION: Additional metabolic and activation assessments of the treated platelets confirmed the previously reported superiority of PAS-IIIM over PAS-III, but extended it to the INTERCEPT™ process.

Efficiency of blood culture bottles for the fungal sterility testing of corneal organ culture media.

BACKGROUND/AIM: The consequences of fungal contamination of an organ cultured cornea, though exceptional, are often disastrous for the recipient. Consequently, eye banks often quarantine corneas for 10 days or more before passing them for grafting. This period, though detrimental to the endothelial cell density of the delivered cornea, is necessary to detect contamination using conventional microbiological methods. The authors previously validated the use of a pair of aerobic and anaerobic blood bottles for sensitive and rapid detection of bacteria. To allow a short quarantine period, it remained only to optimise detection of fungi. The authors aimed to compare sensitivity and rapidity of fungal contamination detection by three methods: blood bottles, Sabouraud, and daily visual inspection of the organ culture medium. METHODS: Four inocula (10(6), 10(4), 10(2), 10 colony forming unit (CFU) per ml) of 11 fungi (Candida albicans, C tropicalis, C glabrata, Saccharomyces cerevisiae, Rhodotorula rubra, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus niger, A fumigatus, A flavus, Acremonium falciforme) were inoculated in a commercial organ culture medium containing a coloured pH indicator (CorneaMax, Eurobio, Les Ulis, France). The real live fungal inoculum was verified immediately after inoculation. After 48 hours at 31 degrees C, samples of the contaminated media were inoculated in three blood bottles: Bactec Aerobic/F, Bactec Mycosis IC/F, and Bactec Myco/F Lytic (Becton Dickinson, Le Pont de Claix, France), then placed in a Bactec 9240 rocking automat, and in four Sabouraud media (solid and liquid, 28 degrees C and 37 degrees C) with daily observation. Contaminated organ culture media were also checked daily for any change in turbidity and/or colour. Experiments were performed in triplicate. RESULTS: Mycosis IC/F and Myco/F Lytic bottles were neither faster nor more sensitive than the aerobic bottle. The three methods were positive for all inocula, even the lowest (viable inoculum below 10 CFU/ml for each fungus). Contamination was detected within 24 hours by the aerobic bottles in 91% (40/44), by Sabouraud in 98% (43/44) (no significant difference) and by visual inspection in 66% of cases (29/44) (p<0.001 with the two others). Maximum times to detection were 46, 48 and 72 hours respectively. CONCLUSION: This study further counters the preconception that fungal contamination is hard to detect in corneal organ culture media. This study is the last step in validating the use of a pair of blood bottles for the sterility testing of organ culture media, this time for fungi. Their use should make it possible to shorten microbiological quarantine and thus deliver corneas with higher endothelial cell density, without increasing the risk of recipient contamination.

Differential downstream effects of CD40 ligation mediated by membrane or soluble CD40L and agonistic Ab: a study on purified human B cells.

With the addition of various cytokines, the CD40-CD40 ligand (CD40L) system can act as a T-helper cell surrogate to permit B lymphocytes to produce large amounts of polyclonal Ig. In the present study, we tested six CD40-CD40L stimulation models: (i, ii) soluble agonistic 89 and G28.5 mAbs ; (iii, iv) 89 and G28.5 bound via their Fc fragments on CDw32-transfected mouse fibroblasts; (v) purified, soluble, trimeric human CD40L molecules (sCD40L); and (vi) human CD40L expressed by a CD40L-transfected mouse fibroblastic cell line (LCD40L). Target B cells consisted of purified blood and tonsillar CD19+ lymphocytes cultured in the presence of CD40 stimuli and IL-2 and IL-10, added at the onset of each B cell culture. A) There was differential expression of CD69, CD80 and CD86 exposure to sCD40L and LCD40L was ensued by the strongest % MFI changes over control. B) In blood B cells, mAbs and sCD40L induced IgA, IgM and IgG production almost equally well; LCD40L proved less efficient. In contrast, in tonsil B cells, LCD40L induced significantly more IgA, IgG1, IgG3 and IgM production than other signals. Using certain CD40/CD40L stimuli to model in vitro Ig production, a system used regularly in many laboratories, may affect the interpretation based on the cell type and on the CD40/CD40L system used.

Is manual counting of corneal endothelial cell density in eye banks still acceptable? The French experience.

AIM: To examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant. METHODS: A questionnaire was sent to the 22 eye banks asking for details of the technical features of the light microscopes used, the microscope calibration, strategy for cell counting, the technical staff, and the method of presenting endothelial data. RESULTS: All eye banks responded and 91% (20/22) used only manual counting methods, in real time, directly through a microscope, and 62 different technicians, with varying experience, were involved in such counting. Counting of cells within the borders of a grid that were in contact with two adjacent borders was the most common method (17/22, 77%). Of the eight banks (8/22, 36%) that did not calibrate their microscopes, six reported the highest ECD values. Of the 14 others (64%), six applied a "magnification correcting factor" to the initial cell counts. In five of these cases, the corrected ECD was lower than estimated on initial count. Most of the banks (12/22, 55%) counted 100 cells or less in one to six non-adjacent zones of the mosaic. 14 of the banks (14/22, 64%) also graded cell polymegethism while seven (7/22, 32%) also graded pleomorphism ("hexagonality"). CONCLUSIONS: Lack of microscope calibration appears to be the leading cause of variance in ECD estimates in French eye banks. Other factors such as differences in counting strategy, the evaluation of smaller numbers of cells, and the different extent of experience of the technicians may also contribute to intraobserver and interobserver variability. Further comparative studies, including cross checking and the outcome of repeated counts from manual methods, are clearly needed with cross calibration to a computer based image archiving and analysis system.

[Optimizing microbiological controls of corneal organ culture media]. / Optimisation du contrôle microbiologique des milieux d'organoculture des greffons cornéens.

BACKGROUND/AIMS: To compare the efficiency of an automated method using blood bottles with conventional microbiological tests for controlling sterility in cornea organ culture media. METHODS: Two complementary studies were conducted. Experimental study: standard organ culture media were contaminated with four different inocula of 14 bacteria and 3 fungi. The bactericidal activity of organ culture media were evaluated after 48 hours of incubation at 31C. Observational study: 357 samples of organ culture media were collected over 1 year in our cornea bank. For both studies, media were inoculated in three blood bottles (aerobic, anaerobic, fungal) placed in an automat with automated detection every 10 minutes, and in three conventional microbiological media as a control. Changes in organ culture medium color and growth on conventional broth were checked daily by visual inspection. All samples were observed experimentally for 14 days. The sensitivity and rapidity of contamination detection were compared across the three methods: blood bottles, conventional method, and visual inspection of medium color. RESULTS: Experimental study: organ culture medium eradicated five bacteria: S. pneumoniae, B. catarrhalis, E. coli, P. acnes and H. influenzae. For the others, (Methicillin-resistant S. aureus, Methicillin-sensitive S. aureus, S. epidermidis, S. haemolyticus, P. aeruginosa, A. baumannii, B. subtilis, K. pneumoniae, E. faecalis, C. albicans, C. kruzei, A. fumigatus) the blood bottle method, the conventional microbiological method, and the visual inspection detected microbiological growth respectively in 100%, 76.5%, and 70% of cases. Mean detection time using blood bottles was 15.1 hours (standard deviation, 13.8; range, 2-52). In cases of detection by the blood-bottle method and the conventional method, the former was always faster: 95.5% versus 65.2% detection within 24 hours (p=0.022). Observational study: the global contamination rate was 8% (29/357 analysis). The gain in sensitivity with blood bottles was 25% compared with the conventional method. Five bacteria (three coag. neg Staphylococcus, one E. faecalis, one P. paucimobilis) were detected only by the blood bottles. In addition, these were always detected more quickly with, respectively, 66.6% versus 26.6% detection with 24 hours (p=0.028). CONCLUSIONS: Blood bottles detect contaminations of cornea organ culture media more efficiently and faster than conventional microbiological methods. They make it possible to reduce the quarantine period with an equally high security level. Consequently, they should be recommended in cornea preservation guidelines.

Sensitivity and rapidity of blood culture bottles in the detection of cornea organ culture media contamination by bacteria and fungi.

AIMS: To test the bactericidal activity of standard organ culture medium, and to compare the sensitivity and rapidity of blood culture bottles with conventional microbiological methods for detection of bacteria and fungi inoculated in a standard cornea organ culture medium. METHODS: The bactericidal activity of contaminated standard organ culture medium containing 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 micro g/ml amphotericin B was evaluated after 48 hours of incubation at 31 degrees C with five inocula of 14 bacteria. Two yeasts (Candida spp) and one Aspergillus were also tested. Contaminated media were then inoculated in three blood bottles (aerobic, anaerobic, fungal) placed in a Bactec 9240 automat; three conventional microbiological broths were the control. Changes in colour of organ culture medium and growth on conventional broth were screened daily by visual inspection. The sensitivity and rapidity of detection of contamination were compared between the three methods: blood bottle, conventional, and visual. RESULTS: Organ culture medium eradicated five bacteria irrespective of the starting inoculums: Streptococcus pneumoniae, Branhamella catarrhalis, Escherichia coli, Propionibacterium acnes, and Haemophilus influenzae. For micro-organisms where the medium was ineffective or bactericidal only (methicillin resistant Staphylococcus aureus, methicillin sensitive Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Acinetobacter baumannii, Bacillus subtilis, Klebsiella pneumoniae, Enterococcus faecalis, Candida albicans, Candida kruzei, Aspergillus fumigatus), the blood bottle, conventional, and visual methods detected microbial growth in 100%, 76.5%, and 70% of cases respectively. Mean detection time using blood bottles was 15.1 hours (SD 13.8, range 2-52). In cases of detection by the blood bottle method and the conventional method, the former was always faster: 95.5% against 65.2% detection within 24 hours (p=0.022) respectively. CONCLUSIONS: Blood bottles detect more efficiently and more rapidly a wider range of bacteria and fungi than the conventional microbiological method and the visual inspection of organ culture media.

[Cornea donation consent by telephone]. / Recueil du consentement au don de cornée par téléphone.

BACKGROUND: The cornea donation process often runs into problems of obtaining family consent. A face-to-face interview is often not possible for logistical reasons. We carried out a prospective study on the effectiveness of telephone contact in obtaining donation consent. MATERIAL AND METHODS: Consent was obtained by a single, non medical, hospital coordinator. He contacted families selected on good staff-family relations during the patient's stay. If a face-to-face interview was not possible, a telephone interview was conducted using a standardized procedure. RESULTS: Over 21 months, 334 families were contacted, either in a face-to-face interview (142, 42.5%) or by telephone (192, 57.5%). Donation consent was obtained in 66.5% of cases, 106 times by telephone (47.7%) and 116 times in the face-to-face interview (52.3%). The acceptance rate was 55.2% by telephone and 81.6% face to face (p<0.001). CONCLUSIONS: The telephone interview was an effective method for obtaining consent for cornea donation. Although the acceptance rate using this method is lower than the face-to-face interview, using the telephone should not be overlooked as this enabled procurement of nearly half the corneas in our hospital.

[Automated analyser of organ cultured corneal endothelial mosaic]. / Mise au point d'un analyseur automatique de l'endothélium des greffons cornéens conservés.

PURPOSE: Until now, organ-cultured corneal endothelial mosaic has been assessed in France by cell counting using a calibrated graticule, or by drawing cells on a computerized image. The former method is unsatisfactory because it is characterized by a lack of objective evaluation of the cell surface and hexagonality and it requires an experienced technician. The latter method is time-consuming and requires careful attention. We aimed to make an efficient, fast and easy to use, automated digital analyzer of video images of the corneal endothelium. METHODS: The hardware included a PC Pentium III ((R)) 800 MHz-Ram 256, a Data Translation 3155 acquisition card, a Sony SC 75 CE CCD camera, and a 22-inch screen. Special functions for automated cell boundary determination consisted of Plug-in programs included in the ImageTool software. Calibration was performed using a calibrated micrometer. Cell densities of 40 organ-cultured corneas measured by both manual and automated counting were compared using parametric tests (Student's t test for paired variables and the Pearson correlation coefficient). RESULTS: All steps were considered more ergonomic i.e., endothelial image capture, image selection, thresholding of multiple areas of interest, automated cell count, automated detection of errors in cell boundary drawing, presentation of the results in an HTML file including the number of counted cells, cell density, coefficient of variation of cell area, cell surface histogram and cell hexagonality. The device was efficient because the global process lasted on average 7 minutes and did not require an experienced technician. The correlation between cell densities obtained with both methods was high (r=+0.84, p<0.001). The results showed an under-estimation using manual counting (2191+/-322 vs. 2273+/-457 cell/mm(2), p=0.046), compared with the automated method. CONCLUSIONS: Our automated endothelial cell analyzer is efficient and gives reliable results quickly and easily. A multicentric validation would allow us to standardize cell counts among cornea banks in our country.

Automated tri-image analysis of stored corneal endothelium.

BACKGROUND: Endothelial examination of organ culture stored corneas is usually done manually and on several mosaic zones. Some banks use an image analyser that takes account of only one zone. This method is restricted by image quality, and may be inaccurate if endothelial cell density (ECD) within the mosaic is not homogeneous. The authors have developed an analyser that has tools for automatic error detection and correction, and can measure ECD and perform morphometry on multiple zones of three images of the endothelial mosaic. METHODS: 60 human corneas were divided into two equal groups: group 1 with homogeneous mosaics, group 2 with heterogeneous ones. Three standard microscopy video images of the endothelium, graded by quality, were analysed either in isolation (so called mono-image analysis) or simultaneously (so called tri-image analysis), with 50 or 300 endothelial cells (ECs) counted. The automated analysis was compared with the manual analysis, which concerned 10 non-adjacent zones and about 300 cells. For each analysis method, failures and durations were studied according to image quality. RESULTS: All corneas were able to undergo analysis, in about 2 or 7.5 minutes for 50 and 300 ECs respectively. The tri-image analysis did not increase analysis time and never failed, even with mediocre images. The tri-image analysis of 300 ECs was always most highly correlated with the manual count, particularly in the heterogeneous cornea group (r=0.94, p<0.001) and prevented serious count errors. CONCLUSIONS: This analyser allows reliable and rapid analysis of ECD, even for heterogeneous endothelia mosaics and mediocre images.

[Use of a pair of blood culture bottles for sterility testing of corneal organ culture media]. / Intérêt de l'utilisation de flacons à hémoculture pour le contrôle de stérilité microbiologique des greffons cornéens.

PURPOSE: To test the effectiveness and rapidity of a pair of blood culture bottles in the diagnosis of bacterial and fungal contamination of corneal organ culture media. MATERIAL: and methods: Seven hundred and sixty one microbiological analysis of storage media (Inosol(R) and Exosol(R), Opsia, Toulouse, France), sampled in all phases of the organ culture at 31 degrees C of 410 consecutive corneas, were analyzed. Each medium was inoculated in a pair of Bactec Plus Aerobic/F(R) and Bactec Lytic/10 Anaerobic/F(R) blood bottles (Becton Dickinson, Cockeysville, MD) and placed in a Bactec 9240 incubator for 14 days at 37 degrees C and in a Sabouraud broth at 20 degrees C. Changes in color or turbidity of storage media were evaluated daily at the corneal bank. Recipients were screened after graft for signs of infection. RESULTS: The overall contamination rate was 2.4% (18/761). Contamination was detected in less than 1 day in 78% (14/18) and in less than 2 days in 94% (17/18). Positivity of the microbiological controls of starting media preceded medium color changes in 10 out of 14 cases. Bactec blood bottles allowed detection of bacteria as well as Candida sp. yeasts. DISCUSSION: The use of a pair of aerobic and anaerobic blood culture bottles is a simple, effective and rapid method for the diagnosis of a wide range of microbiological contaminations of organ-cultured corneas during banking. CONCLUSION: The validation of this protocol will require a prospective study to compare it with the conventional microbiological method.

[Corneal harvesting from donors over 85 years of age: cornea outcome after banking and grafting]. / Prélèvements de cornées après 85 ans: issue des greffons après organoculture et devenir des greffés.

PURPOSE: To study the suitability of corneas from very old donors for graft after organ culture and their clinical and endothelial outcomes in recipients after perforating keratoplasty. METHODS: We stored 419 corneas at 31 degrees C for 13.1 +/- 4 days (mean +/- SD) and then divided them according to donor age: group 1, donors under 85 years of age (n=3 3 0, 79%, 16-84 years old), and group 2, donors over the age of 85 (n=8 9, 21%, 85-100 years old). Endothelial density at the time of harvest and before and after organ culture, rates of suitability for grafting, and clinical and endothelial outcomes of the 196 keratoplasty procedures were compared in a prospective longitudinal study of the 2 groups, with a mean follow-up of 25 months. The corneas were grafted with no pre-established policy on matching with the age of the receiver. Statistical analysis was carried out on SPSS 10.0: Chi(2), Student t test, and Kaplan Meier survival curves. RESULTS: The average age of the donors was 72.1 +/- 16.7 years. The macroscopic aspect of the corneas was judged to be of slightly lower quality in group 2. No statistically significant difference was found in overall suitability for transplantation (group 1, 45% vs group 2, 54%, p=0.17) but elimination for low endothelial density was more frequent in group 2 (67% vs 39%, p=0.001). Cell density at the beginning of organ culture was lower in very old corneas than in younger corneas (respectively, 2116 +/- 368 vs 2 311 +/- 360 cell/mm(2), p=0.002) but no difference was apparent at the end of organ culture (respectively, 2 011 +/- 285 vs 2 090 +/- 296, p=0.12) because very old corneas lost fewer cells than younger ones (respectively, 5.6% vs 10.0%, p=0.001). There was no correlation between donor/receiver age (r=0. 337) but group 1 corneas were slightly more frequently allotted to receivers with normal endothelium (p=0.019). During surgery, the two groups did not differ in terms of the macroscopic aspect of the grafts. In the 196 grafted patients, and without age-matching, overall graft survival (86% vs 79%, p=0.275), visual acuity, and endothelial density (1 194 +/- 469 vs 1098 +/- 545 cells/mm(2), p=0.387) did not differ at the completion of the study. DISCUSSION: The corneas from very old donors were macroscopically of poorer quality and had a lower cellular endothelial density at harvesting, but these differences disappeared after organ culture because of greater cell loss in corneas from younger donors. Selection by organ culture ensures that functional, anatomical, and cellular results are not influenced by very old donor age. CONCLUSION: Considering the aging population in countries with a high standard of living, the techniques available for selecting corneas based on endothelial quality, and the increasing need for corneal grafts, the very old age should not be deemed off-limits for corneal harvesting.

Cornea procurement from very old donors: post organ culture cornea outcome and recipient graft outcome.

AIM: To study the suitability of corneas from very old donors for graft after banking and their clinical and endothelial outcomes in recipients. METHODS: 419 corneas stored in organ culture were divided into group 1, donors under 85 years (330 corneas) and group 2, "very old" donors aged 85 years and over (89 corneas). Endothelial cell density (ECD) before and after organ culture, discard rate before and after storage, and clinical and endothelial outcomes of the 196 penetrating keratoplasties (PKP) (158 in group 1 and 38 in group 2) were compared in a prospective longitudinal study. RESULTS: Initial ECD was lower in group 2 than in group 1 and elimination for low ECD was more frequent in group 2 (respectively 38% v 20.2%, p=0.001). At the end of storage, because very old corneas lost fewer ECs than younger ones (respectively 4.2% v 9.5%, p=0.022), ECD was comparable between the two groups. The corneas of very old donors had a poorer macroscopic appearance at procurement and during surgery. Despite this, in grafted patients, overall graft survival in groups 1 and 2 (respectively 87.4% v 80.6%, p=0.197), visual acuity, and ECD did not differ at completion of the study (mean follow up 25 months). CONCLUSION: This study suggests that endothelial cell count during banking ensures that functional and cellular results of PKPs are not dramatically influenced by very old donor age. Considering Europe's ageing population, the very elderly should not be deemed off limits for corneal procurement.

Use of a pair of blood culture bottles for sterility testing of corneal organ culture media.

AIMS: To test the effectiveness and rapidity of a pair of blood culture bottles in the diagnosis of bacterial and fungal contamination of corneal organ culture media. METHODS: 761 microbiological analyses of storage media (Inosol and Exosol, Opsia, Toulouse, France), sampled in all phases of the organ culture at 31 degrees C of 410 consecutive corneas, were analysed. Each medium was inoculated in a pair of Bactec Plus Aerobic/F and Bactec Lytic/10 Anaerobic/F blood bottles and placed in a Bactec 9240 incubator for 14 days at 37 degrees C and in a Sabouraud broth at 20 degrees C. Changes in colour or turbidity of storage media were evaluated daily at the corneal bank. Recipients were screened post-graft for infectious signs. RESULTS: Overall contamination rate was 2.4% (18/761). Contamination was detected in less than 1 day in 78% (14/18) and less than 2 days in 94% (17/18). Positivity of the microbiological controls of starting media preceded changes medium colour in 10 out of 14 cases. Bactec blood bottles allowed detection of bacteria as well as yeasts. CONCLUSION: The use of a pair of Bactec blood culture bottles appears reliable for the rapid diagnosis of a wide range of microbiological contaminations of organ cultured corneas during banking.

[Epithelial invasion of the corneal button. Good techniques for human corneal harvesting]. / L'invasion epithéliale du bouton cornéen. A propos des bonnes pratiques de prélèvement des cornées humaines.

Corneoscleral excision is the most common harvesting technique used in France before keratoplasty. We report two cases of corneal button epithelial invasion during organ culture caused by an excision that was too small. Two corneas stored for 23 days in organ culture at 31 degrees C had a cellular proliferation on the surface of Descemet's membrane. Cornea button diameters were respectively 11 and 13mm. The epithelial origin of the invasion was confirmed by examining the flat mount of Descemet's membrane by inverted microscopy and standard histopathology. The authors discuss the origin and the potential consequences of the epithelial invasion and emphasize the importance of good corneal harvesting recommendations before keratoplasty.

Reproducible scoring of CFU-GM and BFU-E grown in collagen-based semisolid medium after a short (3 h) training.

Colony counting remains an important source of variation in colony-forming unit-granulocyte-macrophage (CFU-GM) assays performed in methylcellulose or agar. We studied the reliability of colony scoring of CFU-GM assays carried out with collagen, a matrix that allows gel collection on glass slides and in situ cellular morphology. Fourteen slides were exchanged among laboratories, and two rounds of colony (CFU-GM and burst-forming units-erythrocyte [BFU-E]) counting were performed by 11 (first counting), then 8 (second counting) different laboratories, the majority of which had no previous experience of collagen gel cultures and reading. Two-way analysis of variance (ANOVA) of the first round of colony counting showed significant differences among centers in CFU-GM counts (p = 0.023) but not in BFU-E counts (p = 0.163). Coefficients of variation for the 14 slides ranged from 22% to 50% (median 28%) for CFU-GM counts and from 12% to 74% (median 23%) for BFU-E counts. After a 3 h session of collective colony reading attended by members of 8 laboratories, a second round of colony counting was performed. This time, ANOVA showed no significant difference among centers for CFU-GM (p = 0.533) and BFU-E (p = 0.328) counts, and coefficients of variation were significantly improved, with medians of 17% for CFU-GM counts and 20% for BFU-E counts. In addition, when data from the second round of readings were analyzed without the 2 centers counting consistently low (center 8) or consistently high (center 5), variance among centers was further improved for both CFU-GM (p = 0.798) and BFU-E (p = 0.619). In summary, this study shows for the first time that reproducible BFU-E and CFU-GM scoring can be achieved using collagen-based semisolid medium (now commercially available) as long as adequate training in colony identification is provided.

[Can one graft with a positive cross-match?]. / Peut-on greffer avec un cross-match positif?

Differential cross-matches have been proposed to allow immunised patients to be grafted, whereas the dogma of a global positive cross-match discarded them from renal transplantation. We report our one-center experience considering current T positive cross-match as the only contra-indication to grafting, as well as patients whose sera comprise specific anti-donor antibodies. A comprehensive characterization of the antibodies was achieved by identification of auto-antibodies and specification of IgM and IgG isotype, class I and class II specificities, as well as HLA specificities. The differential cross-match comprised an auto and an allo-cross-match, against T and B lymphocytes. Historical and current sera were analysed either untreated or after DTT-treatment, at +4 degrees C and +22 degrees C. We performed 79 renal transplantations across positive cross-matches, which were 20 historical T positive cross-matches, 26 historical B positive cross-matches and 33 current B positive cross-matches. Results and graft survival were strictly identical as those obtained in the transplantations achieved with negative cross-matches throughout the same period, especially in sensitized patients. Current positive B cell cross-matches due to IgG were associated with an increased risk for early graft failure. We conclude that differential cross-match is a safe strategy permitting immunised patients to be grafted.