Sécurité et cytométrie en flux Formation permanente Marseille 23 novembre 2007 Sécurité et cytométrie en flux Gérald Grégori Laboratoire de Microbiologie, Géochimie et Ecologie Marines CNRS UMR 6117 163 Avenue de Luminy - Case 901- Bât TPR1, 13288 Marseille cedex 9 Tél. : (+33) 4.91.82.91.14 Fax : (+33) 4.91.82.96.41 Contact : gerald.gregori@univmed.fr Adapté du Cours de Cytométrie organisé dans le cadre du congrès ISAC (Québec, 2006) par G. Grégori et L. Krebs (Elly Lilly, USA)

Les risques Risques biologiques Risques électriques Irradiation par les LASERS Source: http://www.compliancesigns.com/

Risques biologiques Risques biologiques

Sources communes de risques biologiques en cytométrie en flux Echantillons frais (non fixés) Matériel humain ou animal (singes, porcs) Vecteurs viraux pour transfections génétiques Transmission de maladies entre espèces Pathogènes résistants aux traitement OGM Certains virus carcinogènes (hépatite)

Fausses idées préconçues Le matériel non humain/primate ne présente pas de risque Les échantillons préparés pour les analyses de virus HIV ou Hépatite, & autres maladies sont sûrs Les cellules issues de cultures ne présentent pas de risque Impossible d’obtenir un soutien institutionnel pour améliorer les procédures de manipulation des échantillons

Le risque induit par les aérosols générés lors du tri (buse Jet-in-air) Perfetto et. al., Cytometry A. 2003 Apr;52(2):122-30 Génération de gouttelettes “Normales” Petites (40-200µm) Micro (3-7µm) Le “splash” dans le réceptacle pour la collecte Problème instrumental (bouchage) Tri à haute vitesse / haute pression augmente le risque

Les aérosols : Source de risque élevé Quand risque d’infection potentiel : Niveaux plus élevés de confinement Multiples barrières secondaires pour empêcher le dispersion et la propagation dans l’environnement Biosafety in Microbiological and Biomedical Laboratories Fourth Edition April 1999

Questions sur les risques biologiques ISAC 2005 Surveillance Committee Survey Tri à haute vitesse dans 85% des labos 65% des tris dans labos standards <10% des tris se font en BioSafetyLevel (BSL) 3 67% des labos ne mesurent pas la présence d’aérosols Seulement 21% des tris nécessitent une formation particulière pour du matériel potentiellement à risque Seulement ~ 50% des labos utilisent un questionnaire pour identifier la nature et le risque des échantillons avant d’accepter le tri. These results clearly convey the lack of appropriate safety measures in many sorting facilities J.Lannigan Communication – Ingrid Schmid, UCLA, Chair, ISAC Biosafety Committee

Dépôts potentiels des aérosols Source: www.cdc.gov/niosh/topics/aerosols (Aerosols 101) The head airways region is also called extra thoracic or nasopharyngeal region, and it includes nose, mouth, pharynx and larynx. The lung airways region is also called tracheobronchial region, and it is from the trachea to the terminal bronchioles. In this region, the trachea subdivides into smaller and smaller branches just like an inverted tree. From the trachea to the alveolar surface, there are 23 airway branchings. The pulmonary region is also called alveolar region. This is where gas exchange takes place Droplet nuclei of <5um are able to reach the alveoli of the lung while particles >5um are trapped in the mucous membranes of the airways

Tous les échantillons ont un niveau de risque Risque relatif Tous les échantillons ont un niveau de risque Les manipulations doivent être appropriées au risque Risque faible – échantillons fixés Risque faible à modéré – lignées cellulaires non-humaines/primates et non manipulées (BSL1) Risque élevé – lignées cellulaires humaines/primates, manipulées génétiquement ou cellules infectées (BSL1/2) Risque le plus élevé – matériel primaire humain/primate (BSL2+), cellules infectées (BSL3) In 1994, OSHA issued an interpretation of the applicability of the BBP Standard towards human cell lines. According to the interpretation, human cell lines are considered to be potentially infectious and within the scope of the BBP Standard unless the specific cell line has been characterized to be free of hepatitis viruses, HIV, Epstein-Barr virus, papilloma viruses and other recognized bloodborne pathogens.1 In alignment with this interpretation, the American Type Culture Collection (ATCC) recommends that all human cell lines be accorded the same level of biosafety consideration as a line known to carry HIV.2 Moreover, the Fourth Edition of the CDC publication, Biosafety in Microbiological and Biomedical Laboratories (BMBL), recommends that human and other primate cells should be handled using Biosafety Level 2 (BSL2) practices and containment.3 ATCC quote: Since it is not possible for us to test every cell line for every possible virus, we rely on the tests performed by the depositor. We recommend that all human cell lines be accorded the same level of biosafety consideration as a line known to carry HIV. With infectious virus assays or viral antigen assays, even a negative test result may leave open the possible existence of a latent viral genome.

Eléments de confinement Equipement –Barrière primaire Laboratoire –Barrière secondaire Management du labo & pratique

Equipement de sécurité Equipement personnel de protection Hotte de protection (Biosafety Cabinet (BSC)) Confinement des aérosols

Equipement de protection personnel Vêtements (blouse) Gants Protection faciale (lunettes et masque) Appareil respiratoire

Confinement d’aérosols au niveau du cytomètre Trieur MoFlo™ (DAKO) Trieur FACSAria™ (BD)

Confinement d’aérosols au niveau du cytomètre FACStar Plus™ & FACSVantage SE™ Source: Cytek Development website

Eléments de confinement Equipement –Barrière primaire Laboratoire –Barrière secondaire Protection de l’opérateur Protection des autres membres du laboratoire Protection de l’extérieur du laboratoire Management du labo & pratique Questionnaire sur la nature des échantillons Traçabilité des échantillons Formation du personnel Zones à accès règlementés Bonnes pratiques de laboratoire (pipetage, nettoyage, etc.) Gestion des déchets : javelliser les déchets; fixer les échantillons (Formaldéhyde) Tests/détection de contamination Procédure de décontamination si besoin (Péroxide)

Choc électrique Electrical Shock Source: http://www.compliancesigns.com/

Plaques électriques des trieurs électromagnétiques Courant ~10µA (3X inférieur au seuil de dangerosité humaine) Voltage ~0-4000 V Courant alternatif Source: MoFlo Owners Handbook Source: www.dakousa.com

Choc électrique : Arc électrique des trieurs électromagnétiques Génération d’un arc: résulte de plaques électriques humides Parade: Arrêter l’alimentation des plaques Enlever les plaques et les sécher Remonter les plaques, reprendre le tri Source: www.scienceclarified.com Illustration of electric arc between two metals. Reproduced by permission of Photo Researchers, Inc.

Choc électrique issu de la charge du jet Les trieurs appliquent une charge électrique au jet pour dévier les gouttelettes à trier Charge ~±200 V Courant alternatif Charge au niveau de la chambre de flux Source: www.dakousa.com

Choc électrique au niveau du cristal piézoélectrique Charge sur le cristal Piézoélectrique 135 V AC Source: www.cytopeia.com

Irradiation par les lasers Irradiation Laser Attention Source: http://www.compliancesigns.com/

Le terme “LASER” est un acronyme … Light Amplification by Stimulated Emission of Radiation

All lasers have the same basic design. The active medium contains the atoms that produce laser light by stimulated emission. This can be a solid crystal, a gas, a semiconductor junction, or a liquid. The excitation mechanism is the source of energy that excites the atoms to the proper energy level for stimulated emission to occur. Solid state lasers use optical sources for excitation; gas lasers use electrical excitation. The active medium and excitation mechanism together form an optical amplifier. Laser light entering one end of the amplifier will be amplified by stimulated emission as it travels through the active medium. The optical resonator is a pair of mirrors at the ends of the active medium. These mirrors are aligned to reflect the laser light back and forth through the active medium. The high reflectance mirror has a reflectivity of nearly 100%. The output coupler has a lower reflectance and allows some of the laser light to pass through to form the output beam. The fraction of the light that is allowed to pass through the output coupler depends on the type of laser. Low power lasers usually require most of the laser light to be reflected to keep the stimulated emission process going, and only a few percent can be allowed to pass into the output beam. In very high power pulsed lasers, the output coupler may have a transmission of over 50%.

Lumière monochromatique Lumière généralement monochromatique (longueur d’onde unique) au contraire d’autres sources lumineuses (soleil & ampoules) Lumière émise dans l’UV, le visible, et l’infrarouge Certains lasers émettent plusieurs longueurs d’ondes.

Brûlures (le plus généralement) Irradiation par LASER Brûlures (le plus généralement) … À l’exception de quelques situations particulières les brûlures de la peau sont moins graves que celles de l’oeil

Collimation (Parallélisme) de la lumière Photons des Lasers cohérents (« parallèles ») L’œil focalise la lumière en un point Focalisation des photons  risque potentiel pour la rétine La lentille de l’oeil augmente la puissance par unité de surface (W/cm2) de la lumière des LASERS par ≥100 000 fois

Dommages de l’oeil Thermique oedème, hémorragie Photochimique (lumière bleue & UV) Production de toxines & modifications biochimiques  inflammation, lésions & opacité Photo-accoustique (impulsions courtes et intenses) explosion induite par l’expansion de gaz

The cornea of the eye is the outer layer The cornea of the eye is the outer layer. It is a transparent protein chemically similar to the white of an egg. When long wavelength CO2 laser light strikes the cornea, it heats it much like a hot frying pan heats an egg white. Ultraviolet exposure of the cornea produces a photochemical effect called photokeratitis, also known as welder’s flash. This is a painful but temporary condition. The lens of the eye absorbs ultraviolet light. The long term effect is the formation of scar tissue on the surface of the lens. This is called a cataract. Reducing UV exposure is important in preventing cataracts later in life. Polycarbonate shop glasses block the wavelengths most likely to cause cataracts. The macula is the area of the retina with the greatest concentration of cones for color vision and high visual acuity. Damage to the macula will result in the greatest loss of vision. The fovea is a dip in the center of the macula.

Comment les lasers ciblent les différentes structures de l’oeil < 400 nm 400 - 1400 nm Rétine > 1400 nm Lentille Cornée

Risques majeurs sur la rétine Lumière Laser entre 400-1400nm (risque pour la rétine) intensifiée par 100 000 X par l’effet lentille de l’oeil. Focalisation sur la macula, la région la plus sensible et la plus importante de la rétine. Cécité partielle (lumière directe ou réflexion).

Exemple –Laser Argon  = 488 nm et 514 nm (visible) Laser continu Puissance de sortie = 700 mW Classe 4 Organe ciblé: rétine Dommage: thermique

Exemples de dommages induits par Laser Brûlure sur rétine Source: Laser-Professionals.com

Résumé des longueurs d’onde à risque pour les structures de l’oeil 100 200 315 400 700 1400 3000 UVC UVB UVA VISIBLE IRA IRB IRC Lentille Rétine Lentille Cornée Cornée Photochimique Thermique

Most cytometer lasers are Class I with coverings & interlocks operative A class 1 laser is incapable of causing an injury during normal use. Lasers can be class 1 because they are very low power or because the beam is fully enclosed. The operators of class 1 lasers do not need to take any precautions to protect themselves from laser hazards. The class 1 limits for visible lasers under the 2000 version of the ANSI Standard vary with laser wavelength. Visible lasers with wavelengths longer than 500 nm have a class 1 limit of 0.4 mW. The class 1 limit for visible lasers with wavelengths shorter than 450 nm is 40 mW. Power limits have been increased from the 1993 version because we now know that they had been set lower than necessary for safety. The CDRH class 1 limit is 0.4 microwatts for the entire visible. The power limits have not yet been changed since it took effect in 1976.

Puissance (mW) Lasers continus, Visibles (0.4 - 0.7 µm) Classement des Lasers Classe Description Puissance (mW) Lasers continus, Visibles (0.4 - 0.7 µm) I Pas de danger < 0.5 II Danger si longue exposition 0.5 – 1.0 III Dangereux par lumière directe & réflexion IIIa Idem 1.0 – 5.0 IIIb 5.0 – 500.0 IV idem > 500.0

Class 2 lasers must be visible Class 2 lasers must be visible. The natural aversion response to bright light will cause a person to blink before a class 2 laser can produce an eye injury. The average for a human aversion response to bright light is 190 ms. The maximum aversion time is always less that 0.25 s. The only protection you need from a class 2 laser is to know not to overcome the aversion response and stare directly into the beam. This has been done, and people have burned their retinas doing it.

Class 3a lasers are “Marginally Unsafe Class 3a lasers are “Marginally Unsafe.” This means that the aversion response is not adequate protection for a direct exposure of the eye to the laser beam, but the actual hazard level is low, and minimum precautions will result in safe use. The CDRH Standard (FLPPS) allows only visible lasers in class 3a. The CW power is limited to 5 mW. If the laser has a small beam so that more than 1 mW can enter the pupil of the eye, it carries a DANGER label. If the beam is expanded to be large enough that only 1 mW can pass through the pupil, the laser carries a CAUTION label. The ANSI Standard has the same limits for visible class 3a lasers. It also allows invisible lasers in this class. An invisible laser with 1 to 5 times the class 1 limit is a class 3a invisible laser under the ANSI Standard. The only precautions required for safe use of a class 3a laser are that the laser user must recognize the level of hazard and avoid direct eye exposure.

Class 3b lasers are hazardous for direct eye exposure to the laser beam. But diffuse reflections are not usually hazardous. The maximum power for a CW class 3b laser is 0.5 W. The maximum pulse energy for a pulsed class 3b laser is 0.125 J outside the retinal hazard region. The maximum pulse energy in the visible and near IR varies with the wavelength. For visible lasers the maximum pulse energy is 30 mJ. It increases to 50 mJ per pulse in the wavelength range of 1050-1400 nm. Class 3b lasers operating near the upper power or energy limit of the class may produce minor skin hazards. However, this is not usually a real concern. Most class 3b lasers do not produce diffuse reflection hazards. However, visible or near IR class 3b lasers with short pulses can produce diffuse reflection hazards of several meters. Your laser safety officer will perform a hazard analysis.

Class 4 lasers are powerful enough that even the diffuse reflection is a hazard. The lower power limit for CW and repetitive pulsed class 4 lasers is an average power of 0.5 W. The lower limit for single pulse class 4 lasers varies from 0.03 J for visible wavelengths to 0.15 J for some near infrared wavelengths. Class 4 lasers require the application of the most stringent control measures.

Exemple de lasers sur cytomètres analyseurs-trieurs DAKO MoFlo Laser Options Solid-State Lasers 635nm, 25mW diode 488nm, 200 mW diode Air-Cooled Laser Melles Griot HeNe 25mW, LHP 928 Water-Cooled Lasers from Coherent® Enterprise II 621, 488 + UV Innova 70-2W, Argon Innova 70-3W, Argon Innova 70-4W, Argon Innova 70-5W + UV, Argon Innova 70-5W, Argon Innova 70K, Krypton Innova 70-Spec, Argon Innova 70-Spec + UV Innova 90C-3W Innova 90C-4W Innova 90C-4W + UV Innova 90C-5W Innova 90C-5W + UV Innova 90C-6W Innova 90C-6W +UV Innova 90C-K, Krypton Innova 302-0.6W Krypton + UV Innova 304-4W, Argon Innova 305-5W, Argon Innova 305 + UV Innova 306-6W, Argon Source: www.cytopeia.com Cytopeia inFlux™ V-GS Laser Options Solid-State Lasers UV 355nm Violet 408nm Violet-Blue 479nm Blue-488nm Green-531nm Red-635nm Red-647nm Coherent Water-Cooled 70, 90, 300 series lasers Source: www.dakousa.com

Autres risques LASER Choc électrique (voltage élevé Exposition chimique Matériel cryogénique Gaz comprimé Explosions Contamination de l’air …