6 Safety in the Immunology-Serology Laboratory
7 Quality Assurance and Quality Control 8 Basic Serologic Laboratory Techniques 9 Point-of-Care Testing
10 Agglutination Methods 11 Electrophoresis Techniques
12 Labeling Techniques in Immunoassay 13 Automated Procedures
14 Molecular Techniques
In the immunology-serology laboratory, precautions must be taken to prevent accidental exposure to infectious diseases and other laboratory hazards. Clinical laboratory personnel are routinely exposed to potential hazards in their daily activities.
SAFETY STANDARDS AND AGENCIES
Safety standards for clinical laboratories are initiated, gov-erned, and reviewed by several agencies or committees. These include the following:
• U.S. Department of Labor, Occupational Safety and Health Administration (OSHA)
• Clinical and Laboratory Standards Institute (CLSI), a nonprofit educational organization that provides a forum for the development, promotion, and use of national and international standards
• Centers for Disease Control and Prevention (CDC), part of the U.S. Department of Health and Human Services Public Health Service
• College of American Pathologists (CAP) • The Joint Commission (TJC)
The primary purpose of OSHA standards is to ensure safe the U.S. Department of Labor through OSHA. The pro-grams deal with many aspects of safety and health protection, including compliance arrangements, inspection procedures, penalties for noncompliance, complaint procedures, duties and responsibilities for administration and operation of the system, and how the standards are set. Responsibility for compliance is placed on the administration of the institution and the employee.
OSHA standards, where appropriate, include provisions for warning labels or other appropriate forms of warning to alert all workers to potential hazards, suitable protective equipment, exposure control procedures, and implementation of training disposal of hazardous chemicals. Information is provided by chemical manufacturers and suppliers about each chemical and accompanies the shipment of each chemical. Each MSDS con-tains basic information about the specific chemical or product, including its trade name, chemical name and synonyms, chemi- cal family, manufacturer’s name and address, emergency tele-phone number for further information about the chemical, hazardous ingredients, physical data, fire and explosion data, and health hazard and protection information. The MSDS describes the effects of overexposure or exceeding the threshold limit value of allowable exposure for an employee in an 8-hour day. The MSDS also describes protective personal clothing and equipment requirements, first aid practices, spill information, and disposal procedures.
In 2006, the CDC introduced the National Healthcare Safety Network (NHSN). This voluntary system integrates a inadvertent contamination with blood or body fluids, as follows:
1. Staff must wear laboratory coats and be additionally protected from contamination by infectious agents.
2. Food and drinks should not be consumed in work areas or stored in the same area as specimens. Containers, refrigerators, or freezers used for specimens should be marked as containing a biohazard.
3. Specimens needing centrifugation are capped and placed into a centrifuge with a sealed dome.
4. A gauze square is used when opening rubber-stoppered test tubes to minimize aerosol production (introduction of substances into the air).
autoclaving autodilutor biohazard biosafety policies
enzyme immunoassay (EIA) immunocompromised immunoprophylaxis infectious waste
nonintact
nosocomial transmission occlusive
percutaneous (parenteral)
personal protective equipment (PPE) phlebotomy
pipetting seroconversion
seronegative sharps skin lesions
Standard Precautions Western blot (WB) window period Key Terms
5. Autodilutors or safety bulbs are used for pipetting.
Pipetting of any clinical material by mouth is strictly forbidden.
Each laboratory must have an up to date safety manual. This manual should contain a comprehensive listing of approved policies, acceptable practices, and precautions, including Stan-dard Blood and Body Fluid Precautions. Specific standards that conform to current state and federal requirements (e.g., OSHA regulations) must be included in the manual.
PREVENTION OF TRANSMISSION OF INFECTIOUS DISEASES
According to the CDC concept of Standard Precautions, all human blood and other body fluids are treated as potentially infectious for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other blood-borne microorganisms that can cause disease in human beings. Compliance with the OSHA Bloodborne Pathogens Standard and the Occupational Exposure Standard is required to provide a safe work environ-ment. OSHA mandates that the employer do the following:
• Educate and train all health care workers in Standard Precautions and in preventing bloodborne infections.
• Provide proper equipment and supplies (e.g., gloves).
• Monitor compliance with protective biosafety policies.
Blood is the most important source of HIV, HBV, and other bloodborne pathogens in the occupational setting. HBV can be present in extraordinarily high concentrations in blood, but HIV is usually found in lower concentrations. HBV may be stable in dried blood and blood products at 25° C for up to 7 days. HIV retains infectivity for more than 3 days in dried specimens at room temperature and for more than 1 week in an transmission in the occupational setting is greater for HBV than HIV.
• Concentration of HBV or HIV; viral concentration is higher for HBV than HIV
• Duration of the contact
• Presence or skin lesions or abrasions on the hands or exposed skin of the health care worker
• Immune status of the health care worker for HBV Both HBV and HIV may be directly transmitted by various portals of entry. In the occupational setting, however, the fol-lowing situations may lead to infection:
• Percutaneous (parenteral) inoculation of blood, plasma, serum, or certain other body fluids from accidental needlesticks
• Contamination of the skin with blood or certain body fluids without overt puncture, caused by scratches, abrasions, burns, weeping, or exudative skin lesions • Exposure of mucous membranes (oral, nasal, or
conjunctival) to blood or certain body fluids, as the direct result of pipetting by mouth, splashes, or spattering
• Centrifuge accidents or the improper removal of rubber stoppers from test tubes, producing droplets. If these that the average risk of HIV transmission is approximately 0.3% after percutaneous exposure to HIV-infected blood and 0.09% after mucous membrane exposure.
SAFE WORK PRACTICES FOR INFECTION CONTROL
The use of CDC Standard Precautions is an approach to infec-tion control that prevents occupational exposures to blood-borne pathogens. It eliminates the need for separate isolation procedures for patients known or suspected to be infectious.
The application of Standard Precautions also eliminates the need for warning labels on specimens.
OSHA requires laboratories to have a personal protective equipment (PPE) program. The components of this regulation include the following:
• A workplace hazard assessment, with a written hazard certification
• Proper equipment selection
• Employee information and training, with written competency certification
• Regular reassessment of work hazards
masks would normally not be needed. Standard Precautions are intended to supplement rather than replace handwashing recommendations for routine infection control. The risk of
nosocomial transmission of HBV, HIV, and other blood-borne pathogens can be minimized if laboratory personnel are aware of and adhere to essential safety guidelines.
PROTECTIVE TECHNIQUES FOR INFECTION CONTROL
Selection and Use of Gloves
Gloves for medical use are sterile surgical or nonsterile exam-ination gloves made of vinyl or latex. There are no reported differences in barrier effectiveness between intact latex and intact vinyl gloves. Tactile differences have been observed between the two types of gloves, with latex gloves providing more tactile sensitivity; however, either type is usually satis-factory for phlebotomy and as a protective barrier during technical procedures. Latex-free gloves should be available for personnel with sensitivity to usual glove material. Rubber household gloves may be used for cleaning procedures.
General guidelines related to the selection and general use of gloves include the following:
1. Use sterile gloves for procedures involving contact with normally sterile areas of the body or during procedures in which sterility has been established and must be maintained.
2. Use nonsterile examination gloves for procedures that do not require the use of sterile gloves. Gloves must be worn when receiving phlebotomy training. The National Institute of Occupational Safety and Health mandates the use of gloves for phlebotomy.
3. Gloves should be changed between each patient contact.
4. Wear gloves when processing blood specimens, reagents, or blood products, including reagent red blood cells.
5. Gloves should be changed frequently and immediately if they become visibly contaminated with blood or certain body fluids or if physical damage occurs.
6. Do not wash or disinfect latex or vinyl gloves for reuse.
7. Using items potentially contaminated with human blood or certain body fluids (e.g., specimen containers,
Facial Barrier Protection and Occlusive Bandages Facial barrier protection should be used if there is a potential for splashing or spraying of blood or certain body fluids. Masks and facial protection should be worn if mucous membrane
contact with blood or body fluids is anticipated. All disruptions of exposed skin, including defects on the arms, face, and neck, should be covered with a water-impermeable occlusive bandage.
Laboratory Coats or Gowns as Barrier Protection A color-coded, two–laboratory coat or equivalent system should be used whenever laboratory personnel are working with potentially infectious specimens. The garment worn in the laboratory must be changed or covered with an uncontami-nated coat when leaving the immediate work area. Garments should be changed immediately if grossly contaminated with blood or body fluids to prevent seepage through to street clothes or skin. Contaminated coats or gowns should be placed in an appropriately designated biohazard bag for laundering.
Disposable plastic aprons are recommended if blood or certain body fluids may be splashed. Aprons should be discarded into a biohazard container.
The introduction of water-retardant gowns has been the greatest change in many PPE practices.
HANDWASHING
Frequent handwashing is an important safety precaution. It should be performed after contact with patients and laboratory
1. After completing laboratory work and before leaving the laboratory
2. After removing gloves. The Association for Profession-als in Infection Control and Epidemiology has reported that extreme variability exists in the quality of gloves, with leakage in 4% to 63% of vinyl gloves and in 3% to 52% of latex gloves.
3. Before eating, drinking, applying makeup, and changing contact lenses, and before and after using the bathroom 4. Before all activities that involve hand contact with
mucous membranes or breaks in the skin
5. Immediately after accidental skin contact with blood, body fluids, or tissues
a. If the contact occurs through breaks in gloves, the gloves should be removed immediately and the hands thoroughly washed.
b. If accidental contamination occurs to an exposed area of the skin or because of a break in gloves, wash
1. Take out a glove from its original box 2. Touch only a restricted surface of the glove corresponding to the wrist (at the top edge of the cuff)
3. Don the first glove
4. Take the second glove with the bare hand and touch only a restricted surface of glove corresponding to the wrist I. How to don gloves:
II. How to remove gloves:
5. To avoid touching the skin of the forearm with the gloved hand, turn the external surface of the glove to be donned on the folded fingers of the gloved hand, thus permitting to glove the second hand
6. Once gloved, hands should not touch anything else that is not defined by indications and conditions for glove use
1. Pinch one glove at the wrist level to remove it, without touching the skin of the forearm, and peel away from the hand, thus allowing the glove to turn inside out
2. Hold the removed glove in the gloved hand and slide the fingers of the ungloved hand inside between the glove and the wrist. Remove the second glove by rolling it down the hand and fold into the first glove
3. Discard the removed gloves When the hand hygiene indication occurs before a contact requiring glove use, perform hand hygiene by rubbing with an alcohol-based handrub or by washing with soap and water.
4. Then, perform hand hygiene by rubbing with an alcohol-based handrub or by washing with soap and water.
Figure 6-1 Technique for donning and removing nonsterile examination gloves. (From World Health Organization: Glove use information leaflet, Geneva, Switzerland, 2009, WHO.)
•
• When washing with a nonantimicrobial or antimicro-bial soap, wet hands first with warm water, apply 3 to 5
SPECIMEN-PROCESSING PROTECTION
Specimens should be transported to the laboratory in plastic leakproof bags. Protective gloves should always be worn for handling any type of biological specimen.
Substances can become airborne when the stopper (cap) is popped off a blood-collecting container, a serum sample is poured from one tube to another, or a serum tube is centri-fuged. When the cap is being removed from a specimen tube or a blood collection tube, the top should be covered with a disposable gauze pad or special protective pad. Gauze pads with an impermeable plastic coating on one side can reduce contamination of gloves. The tube should be held away from the body and the cap gently twisted to remove it. Snapping off the cap or top can cause some of the contents to aerosolize.
When not in place on the tube, the cap should still be kept in the gauze and not placed directly on the work surface or countertop.
Specially constructed plastic splash shields are used in many laboratories for the processing of blood specimens. The tube caps are removed behind or under the shield, which acts as a barrier between the worker and specimen tube. This is designed to prevent aerosols from entering the nose, eyes, or mouth.
Laboratory safety boxes are commercially available and can be used for unstoppering tubes or doing other procedures that might cause spattering. Splash shields and safety boxes should be periodically decontaminated.
When specimens are being centrifuged, the tube caps should always be kept on the tubes. Centrifuge covers must be used and left on until the centrifuge stops. The centrifuge should be allowed to stop by itself and should not be manually stopped by the worker.
Another step to lessen the hazard from aerosols is to exer-cise caution in handling pipettes and other equipment used to transfer human specimens, especially pathogenic materi-als. These materials should be discarded properly and carefully.
ADDITIONAL LABORATORY HAZARDS
It cannot be overemphasized that clinical laboratories present many potential hazards simply because of the nature of the work done. In addition to biologic hazards, other hazards in the clinical laboratory include open flames, electrical equip- ment, glassware, chemicals of varying reactivity, flammable sol-vents, and toxic fumes.
In addition to the safety practices common to all laboratory situations, certain procedures are mandatory in a medical labo-ratory. Proper procedures for the handling and disposal of toxic, radioactive, and potentially carcinogenic materials must be included in the safety manual. Information regarding the hazards of particular substances must be included as a safety practice and to comply with the legal right of workers to know about the hazards associated with these substances. Some chemicals (e.g., benzidine) previously used in the laboratory are now known to be carcinogenic and have been replaced with safer chemicals.
Box 6-1 Guidelines for Handwashing and Hand Antisepsis in Health Care Settings
• Wash hands with a nonantimicrobial soap and water or an antimicrobial soap and water when hands are visibly dirty or contaminated with proteinaceous material.
• If hands are not visibly soiled, use an alcohol-based waterless antiseptic agent for routinely decontaminating hands in all other clinical situations.
• Waterless antiseptic agents are highly preferable, but hand antisepsis using an antimicrobial soap may be considered in settings in which time constraints are not an issue and easy access to hand hygiene facilities can be ensured, or in rare cases when a caregiver is intoler-ant of the waterless antiseptic product used in the institution.
• Decontaminate hands after contact with a patient’s intact skin.
• Decontaminate hands after contact with body fluids or excretions, mucous membranes, nonintact skin, or wound dressings, as long as hands are not visibly soiled.
• Decontaminate hands if moving from a contaminated body site to a clean body site during patient care.
• Decontaminate hands after contact with inanimate objects in the immediate vicinity of the patient.
• Decontaminate hands before caring for patients with severe neutropenia or other forms of severe immune suppression.
• Decontaminate hands after removing gloves.
Adapted from Boyce JM, Pittet D; Healthcare Infection Control Practices Advisory Committee; HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force: Guideline for Hand Hygiene in Health-Care Settings. Recommenda-tions of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Society for Healthcare Epidemiology of America/Association for Professionals in Infec-tion Control/Infectious Diseases Society of America., MMWR Recomm Rep 51(RR-16):1–45, 2002.
Table 6-1 Preparation of Diluted Household Bleach Volume
Bleach Volume H2O Ratio Sodium Hypochlorite
1 mL 9 mL 1:10 0.5%
DECONTAMINATION OF WORK SURFACES, EQUIPMENT, AND SPILLS
Sodium hypochlorite solutions are inexpensive and effective broad-spectrum germicidal solutions. Generic sources of sodium hypochlorite include household chlorine bleach. Con- centrations of 1:10 to 1:100 free chlorine are effective, depend-ing on the amount of organic material present on the surface to be cleaned and disinfected. Many chlorine bleaches (available at grocery stores) are not registered by the U.S. Environmental Protection Agency (EPA) for use as surface disinfectants and are unacceptable surface disinfectants. The EPA encourages the use of registered products because the agency reviews them for safety and performance when the products are used according to label instructions. When unregistered products are used for surface disinfection, users do so at their own risk. EPA-registered chemical germicides may be more compatible with certain materials that could be corroded by repeated exposure to sodium hypochlorite, especially the 1:10 dilution.
While wearing gloves, all work surfaces should be cleaned and sanitized at the beginning and end of the shift with a 1:10 dilution of household bleach. Instruments such as scissors or centrifuge carriages should be sanitized daily with a diluted solution of bleach. It is equally important to clean and disinfect work areas frequently during the workday and before and after each shift. Studies have demonstrated that HIV is inactivated rapidly after being exposed to common chemical germicides at concentrations much lower than used in practice. Diluted household bleach prepared daily inactivates HBV in 10 min-utes and HIV in 2 minutes. Disposable materials contaminated with blood must be placed in containers marked “Biohazard”
and properly discarded.
Hepatitis C virus (HCV), HBV, and HIV have never been documented as being transmitted from a housekeeping surface (e.g., countertops). However, an area contaminated by blood or body fluids needs to be treated as potentially hazardous and requires prompt removal and surface disinfection.
Strategies differ for decontaminating spills of blood and other body fluids, based on the setting. The cleanup procedure depends on the porosity of the surface and volume of the spill.
The following protocol is recommended for managing spills in a clinical laboratory:
1. Wear gloves and a laboratory coat.
2. Absorb the blood with disposable towels. Bleach solutions are less effective in the presence of high
2. Absorb the blood with disposable towels. Bleach solutions are less effective in the presence of high