[Transfusion-associated circulatory overload]. / Œdèmes aigus pulmonaires de surcharge post-transfusionnels.

A working group of the French National Hemovigilance Committee has been in charge of heightening awareness of Transfusion-Associated Circulatory Overload (TACO) among physicians and nurses. This multidisciplinary group has produced the present document that focuses on epidemiological data provided by the French haemovigilance network, physiopathology, diagnosis, treatment and specific actions that could prevent or minimize the risk of TACO.

[Factsheets: support in the analysis of recipients' adverse reactions]. / Fiches techniques: aide à l'analyse des effets indésirables receveurs.

In order to help the analysis of adverse effects of transfusion, factsheets have been written by working groups of the French agency for the safety of health products ANSM. Each factsheet deals with a blood transfusion side effect and is composed of five parts, including pathophysiological mechanisms, diagnostic criteria, management recommendations, etiologic investigations and rules for filing the notification form to ANSM. Since 2006, 11 factsheets have been published on the French haemovigilance network website. The major characteristics of the two last sheets published "post-transfusion purpura" and "non erythrocyte incompatibility reaction" are presented, followed by the updated card for "allergy". These factsheets give relevant guidelines allowing better evaluation of recipients' adverse reactions, particularly their diagnosis, severity and accountability. They also could initiate studies among European and international haemovigilance networks.

Hypersensitivity reactions to blood components: document issued by the allergy committee of the French medicines and healthcare products regulatory agency.

These guidelines represent a consensus among experts on hypersensitivity reactions occurring after transfusion of blood components. They cover recognition, investigation, treatment, and prevention of such reactions. Implemented in France under the auspices of the French Medicines and Healthcare Products Regulatory Agency (AFSSAPS) and based on current knowledge, research, and experience, they aim to provide effective and easily teachable means of further improving the quality of hemovigilance databases, promote interest in this field, and help identify possible mechanisms and at-risk patient groups.

[Patient safety and root cause analysis]. / Sécurité des patients et analyse des causes racines.

Safety in the field of transfusion medicine has greatly improved in France. The risk of viral transmission has decreased by a factor greater than 1500 within the last 20 years. In comparison, the risk related to ABO error has decreased only by half. The reporting of critical incidents, which occur at any step of the transfusion procedure is now mandatory in France and is subject to an in-depth analysis, using methods close to that used in aviation safety. The goal of these analyses is to better understand human factors in order to implement more adequate prevention measures.

[Post-transfusion pulmonary oedema: the French hemovigilance network classification method]. / Œdèmes pulmonaires transfusionnels: classification des cas notifiés en hémovigilance.

Pulmonary oedema after transfusion of blood products may be hydrostatic (transfusion-associated circulatory overload [taco]) or exsudative (transfusion-related acute lung injury [trali]). Both conditions have been recognized as major hazards to transfusion recipients. Risk characterization is necessary to improve safety and to monitor trends in the national blood transfusion system. A collaborative multidisciplinary working group of the French National Hemovigilance Committee has proposed an analysis framework for case definitions and classification. The method relies on internationally used definitions and is adapted to the codification procedures used in the french transfusion incident reports electronic data management.

[Hospital blood bank: information system and immuno-hematology]. / Dépôt de sang: apport d'un système informatique dans la sécurité immuno-hématologique.

Due to regulations, hospital blood banks have to equip them with a computer-based information system. This system facilitates the management of the blood bank and ensures the safety of the storage, issuing and traceability of the blood and blood components. It permits to create a medical file for each transfused patient, which contains the characteristics of the blood components transfused and the immunohematological status of the patient, received by electronic data interchange from the blood establishment. Thus, from the assistance to the prescription of blood transfusion to the issuing and traceability of the blood components, the computer-based information system is the guarantee of the transfusion security in a hospital blood bank.

[Operational qualification of the computer-based information system and electronic data interchanges in a hospital blood bank]. / Qualification opérationnelle du système informatique d'un dépôt de sang et de ses échanges de données.

Since 1985, a Council Resolution defines a new approach to technical harmonization and standards. The directives resulting from this new approach establish the essential safety requirements with which products put on the market must conform, and which should therefore enjoy free movement throughout the European Union, owing to a presumption of conformity. However, if the manufacturer is responsible for the conformity of its product in terms of safety, it is the end-user who must make sure that its requirements for a specific use or a considered application are satisfied and that the product meets its needs. It is the objective of the procedure of validation, which calls upon protocols of qualification intended to show the aptitude of a material, a system, a device, of an installation, to meet the requirements of specified quality and safety. This concept of qualification applies to the data-processing software of the hospital blood banks. Only the stage of operational qualification will be developed here. It involves scenarios made up of test files which make it possible to check the electronic data interchanges between the hospital blood bank and the blood establishment (transmission of the results of analysis, transmission of the data of traceability), as well as the functions of assistance to the issuing of the labile blood products.

[Pulmonary complications of transfusion (TACO-TRALI)]. / Complications pulmonaires de la transfusion (TACO-TRALI).

Pulmonary oedemas occurring during or after a blood transfusion appear as the most frequent serious immediate incidents in the French hemovigilance database. They include transfusion-associated circulatory overload (TACO) and transfusion-related acute lung injury (TRALI). TACO are a major cause of transfusion-related death in France. TRALI are more and more recognized and notified. In no case, pooled fresh frozen plasma (100 donations) treated with solvent-detergent were involved in French TRALI cases. A logigrame will allow hemovigilance officers to better classify pulmonary oedemas in e-fit, the French hemovigilance database.

[Supplying surgery blocs in red cell concentrates: a collective approach to improve practices]. / Approvisionnement des blocs opératoires en concentrés érythrocytaires: une démarche collective d'amélioration des pratiques.

The need to adapt red blood cells concentrates management in surgery blocs and resuscitation to the changes of the legal framework has lead to a collective approach to improve practices. Gathered by the regional hemovigilance coordinators of the Drass Ile-de-France (regional office of health and social actions), representatives of doctors' ordering transfusions and hemovigilance correspondents of the Assistance publique-Hôpitaux de Paris and representatives of the EFS (French blood establishment) Ile-de-France, together with representatives of the Afssaps (French health products safety agency), have coordinated an assessment of local transfusion practices in surgery blocs and resuscitation that have to be compliant. Each hospital then offered local improvement actions, approved by regional and national instances. We present this original and collective approach of assessing practices leading to offers that both respond to a legal framework and improve blood products flows without damaging transfusion security.

Detection of rare circulating breast cancer cells by filtration cytometry and identification by DNA content: sensitivity in an experimental model.

Current methods of detecting micrometastases in breast cancer fail in a large proportion of patients. Therefore an improved method for detection of metastases in blood samples could be of great clinical interest both for prognosis and selection of patients for adjuvant systemic therapy. We have developed a new non-invasive method which associates immuno-magnetic separation and filtration cytometry. The sensitivity of our procedure was evaluated in a model system using a mixture from a human breast cancer cell line (MCF-7) and a normal human blood sample. The identification of tumoral cells was achieved by measuring DNA content in comparison with standard cells. The lowest concentration of MCF-7 detected was 1 tumoral cell in 500,000 white blood cells. In addition, filtration cytometry provides a visual control of nuclei permitting the elimination of all doubtful cases and an automatic count of tumoral cells directly per ml of blood, which may be an independent predictor of early relapse. This new method may avoid unnecessary axillary lymph node dissection in patients with negative nodes. Our procedure seems suitable for the detection of rare circulating cells in routine laboratory testing and could be used in other applications.

[CAL 54, a new cell line derived from a human renal carcinoma: characterization and radiosensitivity]. / CAL 54, une nouvelle lignée cellulaire dérivée d'un carcinome rénal humain: caractérisation et radiosensibilité.

A new cell line (CAL 54) was isolated from a malignant pleural effusion of a patient with renal carcinoma. CAL 54 is a continuous and stable cell line. Immunochemical staining showed simultaneous expression of cytokeratin, epithelial membrane antigen and vimentin. Cytometric flow analysis of DNA content reveals one major hyperdiploid population. Histological aspect of the tumor induced in the nude mouse showed well differentiated adenocarcinoma with papillary structure. Radiation response of these cells was evaluated by the colony-forming method and the data were fitted with the linear-quadratic model. Survival at 2 Gy (SF2) was 0.28 and the mean inactivation dose (D) = 1.50 Gy, ranking this cell line among the radiation sensitive cells. CAL 54 may be an informative cell line to investigate radiation effects in the management of renal tumours.

Cell cycle expression of p53 protein, c-Myc gene product and tyrosine-phosphorylation level determined by image analysis in human breast cancer cells.

OBJECTIVE: To investigate the cell cycle expression of p53 protein, c-myc gene product and tyrosine phosphorylation level in human breast cancer cells. STUDY DESIGN: Using a multifluorescence imaging procedure, the concentration per cell in different phases of the cell cycle can be evaluated by analyzing the bivariate contour plot of DNA content versus antigen concentration. RESULTS: Low fluorescence intensity was observed in the G0/G1 phase for the three markers. The analysis of individual cells demonstrated that approximately 10% of cells were negative. During the G1/S transition, the fluorescence intensity of the three antigens increased rapidly. However, after the mild S-phase, the increase of c-myc was more marked than the tyrosine phosphorylation level, whereas p53 protein remained stable, with a slight tendency to decrease. CONCLUSION: This study confirmed that the p53 protein and c-myc gene product could perform a regulatory function in G1/S transition and, consequently, may play an important role in malignant transformation. Like-wise, the variations of tyrosine kinase activity were linked to cellular progression throughout the cell cycle and could be a useful marker of alteration in the growth-factor signaling pathway. Thus, the multifluorescence imaging procedure may provide useful information on the mechanisms of the cell cycle and on malignant transformation.

Cell cycle expression of estrogen receptors determined by image analysis on human breast cancer cells in vitro and in vivo.

We have investigated, by image analysis, the cell cycle expression of estrogen receptors (ER) on MCF-7 cell line and on MCF-7 xenografts. The results demonstrate, in vitro as well as in vivo, an increase of ER concentration during the G0/G1-phase, followed by a decrease during the S-phase until the late S-phase where a rapid increase was noted. These results confirm that estrogens are involved in the DNA synthesis since ER is expressed in vivo at a maximal level in the late G1. In presence of saturating concentrations of 17 beta-estradiol, the mean ER concentration in G0/G1 phase is significantly decreased compared with the control cells cultured in estrogen-deprived medium. This indicates that 17 beta-estradiol down-regulates ER preferentially in the G0/G1 phase. These data suggest that ER in S and G2/M phases is unable to interact with its ligand. Consequently, estrogens may have no effects on the entry of cells in mitosis. Finally, after long-term tamoxifen treatment of MCF-7 xenografts, a tamoxifen-resistant tumor was developed which was characterized by a change in the profile of ER concentration during the G0/G1 phase. In conclusion, it is possible that the differences in cell cycle distribution of ER could be correlated with different phenotypes of breast cancer and also with different clinical phases of tumoral evolution. However, it remains to be known what is the clinical significance of the ER cell cycle expression in relation to tumor aggressiveness and survival.

Cell cycle expression of steroid receptors determined by image analysis on human breast cancer cell line: a hypothesis on the effects of antiestrogens.

Tamoxifen is the widespread anti-hormonal compound used for the treatment of human breast cancer. It is admitted that its effects are mediated via estrogen receptors (ER) but the molecular basis of its activity has yet to be clearly defined. In this work, we have developed a new image cytometry procedure in order to clarify the interactions between steroid receptors and tamoxifen at the cell cycle kinetic level. On untreated cells, an increase of ER level and a decrease of progesterone receptor (PR) level during the G0/G1 phase were demonstrated. Then, the ER and PR levels fell during the S-phase until the beginning of G2/M phase, where an increase was observed, especially for PR. These results suggest that ER is synthesized preferentially during the G0/G1 transition and PR during the S/G2 transition. After short-term tamoxifen treatment an augmentation of ER level was observed which was not dose-dependent, suggesting an increase in receptor translation rather than an augmentation of ER synthesis. PR level declined in the majority of the population leading to a selection of a subset of proliferating PR negative cells after treatment. These data demonstrate that the synthesis of steroid receptors is linked with the progression of cells through the cell cycle and indicate that tamoxifen blocks MCF-7 cells in G1 via its interactions with ER. Our multifluorescence imaging procedure appears to provide a rapid and quantitative approach which is useful for investigating alterations in steroid receptors after endocrine treatment.

Cell growth inhibitory effects of caulerpenyne, a sesquiterpenoid from the marine algae Caulerpa taxifolia.

Caulerpa taxifolia (Vahl) C. Agardh (Ulvophyceae, Caulerpales) is an algae of tropical origin that was accidentally introduced into the Mediterranean in 1984. Caulerpenyne (Cau) is the major metabolite present in Caulerpa taxifolia. This metabolite has previously been shown to be cytotoxic against cell lines in culture as in KB cells and fibroblasts from hamsters. Cau along with 6 other drugs representative of the major classes of anticancer products was tested against 8 cancer cell lines of human origin. Cau demonstrated growth-inhibitory effects in all cases with some variability between cell lines; this inter-cell variability was, however, less marked than that observed with the anticancer drug tested. Cells of colorectal cancer origin were the most sensitive to the presence of Cau with IC50 values of 6.1 and 7.7 microM. Increasing the duration of contact between Cau and the cells from 75 min to 29.5 hr did not improve the cytotoxic efficacy of this compound. When Cau was pre-incubated in the culture medium for from 7 to 83 min before being exposed to CAL 27 cells (head and neck cancer origin), there was a constant loss of cytostatic action of Cau as a function of Cau pre-incubation time. As the bovine serum albumin concentration increased in the culture medium, the concentration-response curves showed a constant shift towards the right, indicating a loss of cytostatic activity of Cau. In the presence of Cau, cells in culture clearly exhibited an early and marked shift into S phase followed by a blockade into the premitotic G2 M phase. Possible targets for CAU remain to be identified. Cau needs to be tested on tumor bearing animals to confirm this promising antiproliferative activity.

Immunohistochemical determination of nuclear antigens by colour image analysis: application for labelling index, estrogen and progesterone receptor status in breast cancer.

The immunohistochemical evaluation of 5-bromo-2'-deoxyuridine (BrdU) labelling index, estrogen (ER) and progesterone receptor (PR) status was carried out on the automated computer-assisted image analysis station BIOCOM 500. Special software has been developed to measure nuclear antigens using the immunoperoxidase method with the Harris hematoxylin counterstain. The analysis was based on the different light adsorption spectra of the chromogen diaminobenzidine and Harris's hematoxylin coloration when exposed to light of differing wavelengths. The results obtained by image analysis were compared to previously validated methods. The thymidine labelling index performed by manual procedures and BrdU incorporation performed by image analysis were comparable (linear correlation coefficient r = 0.76, P < 0.001). Comparison of image analysis and dextran coated charcoal assay for ER and PR content revealed excellent sensitivities and specificities (linear correlation coefficient r = 0.87, P < 0.001 for ER and r = 0.93, P < 0.001 for PR). These data suggest that automated image analysis offers a reliable and reproducible procedure for measuring nuclear antigens.

Effects of tamoxifen on potential doubling time of human breast cancer cell line determined by image cytometry of double fluorescent BrdU and DNA labeling.

Tamoxifen is extensively used for the treatment of human breast cancer. However, the mechanisms by which antiestrogens regulate the growth of estrogen receptor positive tumors have not been totally defined. A new methodology, using automated image analysis BIOCOM 500, was developed for determining potential doubling time (Tpot) of tumors. This new method was checked on three different human breast cancer cell lines (MCF-7, CAL 85-1, CAL 148) in comparison with flow cytometry and then applied to determine the effects of short-term tamoxifen treatment on Tpot of MCF-7 cells. Using the resulting bivariate contour plot of blue fluorescence (DNA content) versus green fluorescence (Bromodeoxyuridine content), a labeling index (LI) value of 0.39 +/- 0.05 and a Tpot value of 21 +/- 2.09 hours were determined for MCF-7 cells. As expected, data demonstrated that 72 hours of 1 microM tamoxifen treatment decreased the LI to 35% by increasing the proportion of G0/G1 cells. It increased the Tpot to 35% compared to untreated cells (Tpot = 31.8 + 4 hours) by a lengthening of G0/G1 phase without changing the length of S phase (Ts = 10.2 +/- 1 hours). At suprapharmacological concentrations (5, 10 microM), an approximately 50% increase in Tpot was observed without modification in Ts. These data suggested a specific cell cycle action of tamoxifen which was probably mediated by mechanisms other than estrogen inhibition, since these experiments were performed in estrogen-deprived medium. In addition, the automated imaging procedure appears to provide a rapid and quantitative approach to determine Tpot in fine needle biopsies which is useful for investigating alterations in cell growth after endocrine treatment or chemotherapy.

Inherent radiosensitivity and split-dose recovery in plateau-phase cultures of 10 human tumour cell lines.

The radiation response of 10 human tumour cell lines, eight of them established in our Institute, was analysed. Single dose acute survival curves were constructed and fitted with the linear-quadratic (LQ) model. The mean inactivation dose (D) was also calculated, together with D(o) and n. In order to measure both split-dose recovery and delayed plating recovery of plateau-phase cells, confluent cultures were subjected to two doses of 2 Gy 24 h apart, and plated 24 h later, simulating clinical fractionation. Survival of these cells (S2 x 2) was compared to that following 4 Gy given to cells plated at low density and an overall recovery factor (RF) was derived, including both types of recovery. S2 x 2 and RF were compared to the intrinsic radiosensitivity parameters. The three melanoma cell lines differed in radiation response, and the three squamous cell carcinoma cell lines were rather radioresistant. The neuroblastoma cell line was highly sensitive, yet it expressed the highest beta and the highest RF. Using a non-parametric correlation test, S2 x 2 was found to be related to D, whereas RF was not related to the radiosensitivity parameters. However, the two cell lines with the lowest D and the lowest S2 expressed the highest RF. These results suggest that radiosensitive cell lines may have a considerable capacity to recover if confluent cultures are exposed to fractionated irradiation. The overall recovery factor (RF) used here is proposed as a useful measure of cellular recovery.

Vitiligo-like depigmentation and morpheas after specific intralymphatic immunotherapy for malignant melanoma.

We report the case of a patient with stage 2 malignant melanoma (MM), who received a specific immunotherapy consisting of intralymphatic injections of irradiated MM cells. She developed subsequently vitiligo-like leukoderma and several plaques of localized scleroderma. Simultaneously an increase in the patient's cytotoxic activity against MM cells was detected in vitro. The responsibility of immune phenomena and specific immunotherapy for the appearance of depigmentation and morpheas is discussed.

A new epithelial membrane antigen (Calam 27) as a marker of carcinoma in serous effusions.

A new monoclonal antibody (Calam 27) that reacts with a membrane antigen present on cells of epithelial origin, but not on cells of mesothelial origin, was investigated as a means of distinguishing between mesothelial cells and malignant cells in cytologic smears of serous effusions from patients with carcinoma. Immunofluorescence staining of cells in 151 effusions from 109 patients with different diseases showed a good correlation between the cytologic diagnosis on routine preparations and the staining with Calam 27. Calam 27 was also used to study the ploidy and cell cycle kinetics of carcinoma cells versus reactive mesothelial cells and normal cells by flow cytometry; these experiments confirmed that Calam 27 is not reactive with mesothelial cells. In conclusion, Calam 27 staining can help to confirm the cytodiagnoses in cases with carcinomatous effusions.