Clinical specimens Body fluids
Human/animal tissues infected with HIV
Growing HIV at research lab scale
Growing HIV-producing cell lines
Working with concentrated HIV preparations
Droplet/aerosol production a
Refer to text for references (BL = biosafety level).
Reports issued by CDC (91) in May 1987 documented that laboratory workers and other clinical staff were occupationally infected with HIV via nonintact skin and mucous membrane exposures. Because the HIV serostatus of the patient sources were unknown at the time of exposure and the exposures were nonparenteral, the CDC issued the "universal blood and body fluid precautions" recommendations in August 1987 (6). The major premise involved the careful handling of all blood and body fluids as if all were contaminated with HIV, HBV, or other bloodborne pathogens. This "universal precautions" concept formed the basis for all subsequent recommendations from CDC (109, 134) and other professional organizations such as the National Committee for Clinical Laboratory Standards (NCCLS) (161).
The counterpart of universal precautions in a laboratory situation involves the consistent use of biosafety level 2 facilities and practices as outlined in the CDC/NIH manual, Biosafety in Microbiological and Biomedical Laboratories (110). The biosafety level 2 precautions are most appropriate for clinical settings or when exposure to human blood, primary human tissue, or cell cultures is anticipated. Standard microbiological practices form the basis for biosafety level 2 with additional protection available from personal protective equipment (PPE) and biological safety cabinets (BSCs) when appropriate.
In 1988, two reports of research laboratory workers with documented occupational HIV infection prompted an investigation by an expert team to review possible sources of exposure and any need to revise current practices to reduce hazards in the research laboratory (99). Subsequently, an agent summary update was issued and included in the 1988 edition of Biosafety in Microbiological and Biomedical Laboratories (162). The expert team did not advise alteration of the CDC/NIH biosafety recommendations for laboratories, but stressed the need for reinforcement of safety practices through proficiency and administrative discipline.
In addition to the advisory nature of the CDC/NIH guidelines, the federal Occupational Safety and Health Administration (OSHA) issued a final standard to regulate occupational exposure to bloodborne pathogens (163). The standard builds on the implementation of "Universal Precautions" specifying the need for control methods, training, compliance, and recordkeeping.
OSHA also recognized that employees in HIV/HBV research laboratories and production facilities may be placed at a higher risk of infection following an exposure because of the concentrated preparations of viruses. Requirements for practices and special procedures, facility design, and additional training for these workplace situations are included in the OSHA standard and are consistent with the CDC/NIH laboratory biosafety guidelines for biosafety levels 2 and 3. 7.1 Specific Precautions
OSHA has issued the bloodborne pathogen standard as a "performance" standard. In other words, the employer has a mandate to develop an exposure control plan to provide a safe work environment, but is allowed some flexibility in order to accomplish this goal. OSHA embraces the basic philosophy of the CDC "universal precautions," and marries it with combinations of engineering controls, work practices, and personal protective equipment in order to accomplish the intent of the standard.
Recognizing the risks inherent in needlestick and sharps exposures, OSHA recently (at time of writing) introduced the Healthcare Worker Needlestick Injury Prevention Act of 1999 (164). The bill would amend the federal Bloodborne Pathogens Standard to require that employers utilize needleless systems and devices with engineered sharps protections to prevent occupational exposure to bloodborne pathogens. It also enhances needlestick reporting requirements and establishes a national clearinghouse to collect data on safety devices. Four states have already adopted revised bloodborne pathogens regulations requiring the use of safety devices as of August 1999: California, Maryland, Texas, and Tennessee.
Exposure control plans for laboratories must adhere to the rules of the OSHA standard, but can also benefit from safety recommendations from other professional organizations such as CDC, NIH, or NCCLS (National Commitee for Clinical Laboratory Standards). Specific rules and recommendations that might augment a laboratory safety plan are worth noting.
7.1.1 Sharps Precautions Since injuries from contaminated sharps represent the highest risk for HIV transmission, clinical and research laboratory safety plans should restrict the use of needles and other sharp instruments in the laboratory for use only when there is no alternative, such as performing phlebotomy. For laboratory procedures, other means should be considered to achieve the job, such as the use of blunt cannulas or small-bore tubing. If needles must be used, investigate the use of "self-sheathing" needles or "needleless" systems that have recently been designed to prevent needlesticks.
Used needles should never be bent, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they should be carefully placed in conveniently located puncture-resistant containers (110). Removal of needles from nondisposable vacutainer sleeves or syringes should be accomplished with a mechanical device such as forceps or hemostats, or by using notched slots designed into needleboxes for safe removal of the needle. The OSHA standard allows a "one-handed" recapping technique only if there is no alternative feasible. All disposable sharps encountered in the laboratory, including pipettes, microtome blades, micropipette tips, capillary tubes, and slides, should also be carefully placed in conveniently located puncture-resistant containers for disposal. Nondisposable sharps should be placed in a hard-walled container for transport to a processing area.
Plasticware should be substituted for glassware whenever possible. Broken glassware should never be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Cotton swabs can be used to retrieve small slivers of glass.
7.1.2 Engineering Controls Recognizing that human behavior is inherently less reliable than mechanical controls, OSHA advocates the use of available technology and devices to isolate or remove hazards from the worker. The use of "self-sheathing" needles is an example of an engineering control to help isolate the worker from the hazard of needlestick exposure.
Another engineering control in the laboratory is the use of a properly maintained biological safety cabinet (BSC) to enclose work with a high potential for creating aerosols or droplets, namely, blending, sonication, necropsy of infected animals, intranasal inoculation of animals, or opening lyophilized vials under pressure. All work with infectious material in an HIV research laboratory should be performed in a BSC or other physical containment device. For example, high energy activities such as centrifugation that are performed outside a BSC should be designed for aerosol containment. Sealed safety cups or rotors should be used for centrifugation, and changed out in a BSC. Before centrifugation, tubes should be examined for cracks, and any glass fragments in the centrifuge cups should be carefully removed with forceps or hemostats. Microwell plate lids can be sealed with tape or replaced with adhesive-backed Mylar film prior to centrifugation.
Plastic shielding can be used to reduce the exposure to splatter or droplets from fluorescent activated cell sorters or other automated laboratory equipment that might generate droplets of infectious material. Likewise, the plexiglass radiation shield used in reverse transcriptase assays offers protection from splatter. However, if used in a BSC, the sloped top may divert airflow in the cabinet, and must be removed to provide optimal protection by the BSC.
High speed blenders and grinders are available that contain aerosols of infectious material, and need to be opened in a BSC after processing. Enclosed electrical incinerators are preferable to open Bunsen burner flames for decontaminating bacteriological loops to prevent splatter, and may be used within or outside a BSC.
7.1.3 Work Practice Controls The manner in which a task is performed can minimize the likelihood of exposures in the laboratory. For example, careful disposal of used needles without recapping or otherwise manipulating by hand can reduce the likelihood of needlesticks.
Standard microbiological practices have been recommended by CDC and NIH guidelines for all laboratory containment levels (110). Most of the practices are designed to prevent indirect transmission of infectious material from environmental surfaces to the hands, and from hands to the mouth or mucous membranes. Such practices include prohibition of mouth pipetting, eating, drinking, smoking, applying cosmetics, or handling contact lenses in the laboratory, and attention to environmental decontamination.
One of the best work practices for any laboratory setting is that of frequent and adequate handwashing when hands are visibly contaminated, after completion of work, before leaving the lab, after removing gloves, and before eating, drinking, smoking, or changing contact lenses. Any standard handwashing product is adequate, but products should be avoided that disrupt skin integrity. When knee- or foot-pedal-controlled faucets are not available, faucets should be turned off by using paper towels used to dry hands to prevent recontamination of hands. Proper attention to handwashing will prevent inadvertant transfer of infectious material from hands to mucous membranes.
In clinical settings, skin lesions may be covered by occlusive dressings and, if lesions are on the hands, gloves worn over the dressings to prevent contamination of nonintact skin. However, workers with skin lesions or dermatitis on hands or wrists should not perform procedures with concentrated HIV material even if wearing gloves. Other work practices can reduce the amount of splatter from laboratory procedures. Covering pressurized vials with plastic-backed or alcohol-soaked gauze when removing needles or when removing tops of pressurized vacutainer tubes will minimize the exposure to splatter. To prevent popping stoppers on evacuated tubes or vials, blood should never be forced into the tube by exerting pressure on the syringe plunger; rather, tubes and vials should be filled by internal vacuum only. Extreme caution should be used when handling pressurized systems such as continous-flow centrifuges, apheresis, or dialysis equipment. Use of imperviously backed absorbent material ("lab diapers") can reduce the amount of splatter on laboratory work surfaces when liquids accidentally leak or fall during lab procedures and can aid in laboratory cleanup. Remember to keep the air-intake and exhaust grilles in BSCs clear of any surface covers or equipment.
Safe transport of specimens or infectious material within the laboratory or to other areas can minimize the potential for accidental spills or injuries. Specimens should be contained in a closed, leakproof primary container, and placed in a secondary container (i.e., a plastic bag) to contain leaks during transport. OSHA regulations do not mandate labeling or color-coding specimens if the specimens are handled only within the facility, a policy implementing "universal precautions" is in effect, and the containers are recognizable as human specimens. Bulk samples may be safely transported in a rack within a sealable plastic container such as a modified "tackle box." The box may need to be labeled with a biohazard symbol or color-coded if the contents are not clearly visible as specimens. Luer caps should be used to transport syringes (needles removed with forceps or hemostats and properly disposed) or needles carefully recapped using a one-handed technique. Capillary tubes should be transported in a solid-walled secondary container such as a screw-top test tube. Transport of cultures or hemocytometers from the BSC within the laboratory may be facilitated by placing them on a tray to limit the number of trips and opportunities for spillage.
Designation of "clean" versus "dirty" areas of the laboratory or within BSCs can help to prevent inadvertent contamination. Work should be planned to move from clean areas to dirty areas. Routine cleaning of work surfaces must be done after procedures are completed and at the end of each workshift, with additional decontamination as needed for spills. Routine cleaning can be accomplished using a variety of disinfectants, including iodophors registered as hard-surface disinfectants, phenolics, and 70% ethanol [with consideration given to the need for longer contact time when decontaminating dried viral cultures (165)]. Diluted bleach has been most widely used for routine disinfection [10% bleach (0.5% sodium hypochlorite) for porous surfaces and 1% bleach (0.05% sodium hypochlorite) for cleaned, hard, and smooth surfaces]. Aldehydes are not recommended for surfaces because of their potential toxicity.
Prompt decontamination is important following spills of infectious materials, since HIV is able to survive for several hours in the environment (see Table 20.1). Appropriate spill cleanup in a clinical setting should involve the following steps:
1. Absorb the spill with towels or "lab diapers" to remove the extraneous organic material.
3. Decontaminate with an appropriate disinfectant [CDC recommends an EPA-registered "hospital disinfectant" that is also "tuberculocidal" or, a 1-10% bleach solution is sufficient (6)].
Large spills of cultured or concentrated agents may be safely handled with an extra step:
1. Flood the spill with an appropriate disinfectant or absorb the spill with granular material impregnated with disinfectant.
2. Carefully soak up the liquid material with absorbent material (paper towels), or scrape up the granular absorbent material and dispose of according to the waste-disposal policy.
3. Clean the area with soap and water.
Laboratory equipment (analyzers, centrifuges, pipettors) should be checked routinely for contamination and appropriately decontaminated. Any equipment sent for repair must also be decontaminated before leaving the laboratory, or labeled as to the biohazard involved.
Because the intent of the OSHA Bloodborne Pathogen Standard is worker protection, the rules for appropriate waste disposal emphasize adequate packaging. Sharps disposal containers must be puncture- and leakproof as well as easily accessible. Other "infectious" or "medical" waste must be placed in leakproof containers or bags that are color-coded red or orange, or labeled with the word "biohazard" or the universal biohazard symbol. All disposal containers should be replaced before they are full.
Blood or body fluids may be disposed of by carefully pouring down the sanitary sewer if local health codes permit, but not poured into a sink where handwashing is performed. Liquid and solid culture materials, however, must be decontaminated before disposal, most commonly by steam sterilization
(autoclaving). Tissues, body parts, and infected animal carcasses are generally incinerated. All laboratory waste from HIV research-scale laboratories or production facilities and animal rooms must be decontaminated before disposal (biosafety level 3 practices). Additional "medical" or "infectious" waste definitions and requirements may exist locally and must be consulted for proper disposal policies.
7.1.4 Personal Protective Equipment (PPE) Another strategy to minimize worker exposure to infectious material is the use of PPEs that are appropriate for the laboratory procedure and the type and extent of exposure anticipated. Examples include a variety of gloves, gowns, aprons, and face, shoe, and head protection. Personal protective equipment may be used in combination with engineering controls and/or work practices for maximum worker protection.
Gloves are required by OSHA when hand contact with blood, other potentially infectious materials, mucous membranes, or nonintact skin is reasonably anticipated. The federal regulations also require gloves when handling or touching contaminated items or surfaces, and for performing vascular access procedures. Gloves are appropriate in the laboratory when handling clinical specimens, infected animals, or soiled equipment, when performing all laboratory procedures in research laboratories, cleaning spills, and handling waste.
For routine procedures, vinyl or latex gloves are effective when appropriately used for prevention of skin exposure to infectious materials. Gloves are not intended to prevent puncture wounds from needles or sharps. However, evidence of a "wiping" function exists that may reduce the amount of blood or infectious material exposure from the outside of the needle as it penetrates a glove or combination of gloves. Johnson et al. (166) found that two or three layers of latex gloves appeared to reduce the frequency of HIV-1 transfer by surgical needles to cell cultures. They also found that Kevlar gloves (untreated), Kevlar gloves (treated with the virucidal compound, nonoxynol-9), and nonoxynol-9-treated cotton gloves used as intermediate layers between two layers of latex gloves significantly reduced the amount of HIV-1 transfer when compared with a single latex glove barrier. Gerberding et al. (167) reported that when surgeons wear double gloves, the rate of puncture of the inner glove is 3 times less than the rate of puncture of a single glove.
Other gloves are available that provide puncture "resistance" such as stainless-steel mesh (chain mail) gloves to protect against injury from large sharp edges such as knife blades. Nitrile gloves (synthetic rubber) have some degree of puncture resistance that may eliminate problems with rings or fingernails, yet retain the necessary dexterity required for performing laboratory procedures. A thin leather glove has been developed that can be worn under latex gloves for an an additional barrier against needlesticks or animal bites. Even heavyweight utility gloves (dishwashing gloves) provide extra protection and should be worn when the procedure permits, such as cleaning contaminated equipment or spills.
Undetected physical holes and leaks require that gloves be frequently inspected and changed. The U.S. Food and Drug Administration (FDA) has issued acceptable quality limits (AQLs) for defects at 2.5% defective for surgeons' gloves and 4.0% for latex exam gloves (168), although the AQL varies widely among manufacturers. The reported percentage defective due to holes for nonsterile latex gloves ranges from 0 to 32%; for nonsterile vinyl gloves, from 0 to 42% (161). Clearly, for high risk situations such as gross contamination of gloves with blood, bloody body fluid, or high concentrations of HIV-1, the use of double gloves will lower the risk of hand contamination from seepage through undetected glove defects. Although they are more puncture-resistant, nitrile gloves are designed to tear apart when any pressure is applied to a hole in the glove, so that any violation of the glove will be detected.
Gloves must never be washed or disinfected for reuse. Detergents may cause enhanced penetration of liquids through undetected holes causing a "wicking" effect (134, 161). Disinfectants, such as 70% ethanol, can also enhance the penetration of the glove barrier and facilitate deterioration (161, 169).
Gloves must be changed when visibly contaminated, torn, or defective, or when tasks are completed. Since hands may be inadvertently contaminated from laboratory surfaces, gloves should be removed before handling telephones, doorknobs, or "clean" equipment. Alternatively, "dirty" equipment may be designated and marked to be handled only with gloved hands. Laboratory workers should practice the aseptic technique for glove removal, specifically, the contaminated side remains on the inside as gloves are removed to protect the worker from skin contamination. Hands should always be washed after glove removal.
When soiling of clothing is anticipated, laboratory coats, gowns, or aprons are recommended. However, when a potential for splashing or spraying exists, solid-front, fluid-resistant gowns are appropriate. If the anticipated exposure involves soaking, solid-front fluidproof gowns are required, as well as hoods/caps, facial protection, and shoe covers. Biosafety level 3 practices advise a solidfront or wraparound, long-sleeved gown or coveralls for adequate protection in research laboratories or production facilities.
Gowns with tightly fitting wrists or elasticized sleeves should be worn for work in BSCs. Alternatively, water-resistant "gauntlets" that provide a barrier between the glove and the laboratory coat are available to reduce skin exposure of the wrist and arm.
Laboratory coats or gowns should not be worn outside the laboratory. In HIV-1 research laboratories or production facilities, the gowns or other protective clothing must be decontaminated before laundering or disposal (BSL-3 practices).
When splashing of blood or infectious material into the mucous membranes of the face is anticipated, a mask and goggles or faceshield must be used. Most laboratory procedures involving this degree of exposure should be conducted within containment equipment such as a BSC. Face protection might be needed for activities conducted outside a BSC, such as performing an arterial puncture, removing cryogenic samples from liquid nitrogen, or in some animal care areas. Masks and eye goggles or faceshields also serve a passive function as a means of preventing accidental contact of contaminated gloved hands with the eyes, nose, and mouth during the course of work activities.
Whatever the PPE needs of any particular laboratory, OSHA requires that the employer provide an adequate supply of PPEs in the appropriate sizes. Hypoallergenic gloves must be available for employees who develop allergies to glove material or the powder inside gloves. Any defective PPE must be replaced, and reusable protective clothing must be laundered and maintained by the institution. Finally, all laboratory workers must be instructed in the proper use of PPEs and their location.
One of the most important components of an exposure control plan for the laboratory is a formal training program. "On-the-job training" is not acceptable as adequate safety training in the laboratory. The recommendations from CDC (109) and NCCLS (161) that emphasize education of laboratory workers have been incorporated into the OSHA bloodborne pathogen standard (163).
Interactive training sessions must be conducted on initial hire and with annual updates by a person knowledgeable about the standard. Employees must be educated regarding their risks and the institution's plan to control these risks.
Recognizing the increased risk of working with concentrated viral preparations, OSHA requires that employees in HIV research laboratories and production facilities receive additional initial training. Employees in these situations must demonstrate proficiency in standard microbiological practices as well as practices and techniques specific to the facility prior to work with HIV. This might include prior experience in handling human pathogens or tissue cultures, or participation in a training program with a progression of work activities to develop proficiency before pathogens are handled.
Employers must ensure compliance with the OSHA standard. The CDC (109) and NCCLS (161) recommend that workplace practices be monitored at regular intervals by a biosafety expert. The NCCLS suggests that audits be conducted that evaluate the existence and effectiveness of training programs and the job descriptions of the safety trainers. The audit should also examine the adequacy of the laboratory facilities and equipment, the standard operating practices, and the written safety protocols. Corrective measures should be implemented if needed. If breaches in protocol are detected, employees should be reeducated and, if necessary, disciplinary action taken.
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