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G U I D E L I N E S T O A V O I D P E R S O N N E L C O N T A M I N A T I O N BY I N F E C T I V E

IN R E S E A R C H L A B O R A T O R I E S T H A T U S E H U M A N T I S S U E S

AGENTS

William E. Grizzle' and Sarah S. Polt

Department of Pathology and the Comprehensive Cancer Center, University of Alabama at


Birmingham and Veterans Administration Medical Center, Birmingham, Alabama 35294

SUMMARY: The personnel in laboratories that utilize tissue and fluids from humans and other primates are at
risk for infection with agents, including the viruses causing hepatitis, AIDS, and other infective agents such as
mycobacteria tuberculosis. To minimize the chance of infection of laboratory personnel, carefully organized
policies and procedures to minimize exposure to infective agents must be established in research laboratories. We
outline some of the approaches of hospital clinical laboratories which have proved most effective in minimizing
transmission of infections from samples to laboratory personnel. Also, we discuss simple considerations important in the use and in the selection of safety equipment. These guidelines and references to other safety information are provided to aid research laboratories in establishing safety procedures that will minimize chances
of personnel contamination with infective agents from research samples.

Key words: laboratory safety; infected human samples; hepatitis; AIDS; HIV; laboratory personnel.
I.

this requirement, the guidelines for the Clinical


Laboratory at the University of Alabama at Birmingham were modified and made more relevant to
research applications. These requirements had been
developed over several years as general protective
measures for dealing with human tissues and fluids.
They had been gleaned from multiple sources, including
publications of laboratory accreditation agencies ~e.g.,
College of American Pathologists and National
Committee for Clinical Laboratory Standards [NCCLS]) and governmental safety regulations.
In preparing these guidelines, we realized that
specific safety" measures to prevent infective contamination were not described in the research literature;
thus, we decided to report our safety guidelines to aid
laboratories that were unaware of those guidelines
provided by the Cooperative Human Tissue Newtwork.
It must be emphasized that these laboratory safety
procedures represent guidelines only. Research investigators must keep themselves informed of the
constantly changing information available on common
infections such as hepatitis and AIDS as well as the
more rare infections such as Creutzfeidt-Jakob disease.
We make no warranty either expressed or implied that
the guidelines discussed herein are adequate to protect
research workers from infection by such agents. The
ultimate responsiblity for laboratory safety lies with the
principal investigator of the laboratory; however, all
research personnel must utilize the safety information
available and maintain responsibility for protecting
themselves and their colleagues. In this regard we
recommend that all research investigators and support

INTRODUCTION
With the accelerated utilization of human tissue in
biological research and with the spread of viral infections within our population, there is an increased risk
of transmission of infection to workers who come in
contact with human tissues and fluids. Personnel
working with human tissues and fluids in clinical settings have been informed of these dangers, and clinical
laboratory workers in hospitals usually have access to
extensive guidelines concerning protective measures
necessary to avoid the transmission of viral and other
infections 11-231.
In contrast, research workers using human tissues are
frequently uninformed as to precautions for avoiding
contamination with infective agents; yet many human
tissues may be obtained from patients with asymptomatic viral or other infections. The literature contains
some general discussions of laboratory safety to prevent
infection with transmissible agents from human tissues.
However, specifics of protective measures are not
described in detail, and most guidelines are published in
clinically related journals.
When the National Cancer Institute funded the
Cooperative Human Tissue Network to increase the
supply of human tissues available for cancer research, it
was mandated that safety guidelines be established for
investigators who received human tissues. In meeting

To whom correspondence should be addressed at Cooperative


Human Tissue Network, Department of Pathology, University of
Alabama at Birmingham, Birmingham, AL 35294.
Journal of Tissue CultureMethods Vol. 11, No. 4, 1988

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1988TissueCultureAssociation,Inc.

GRIZZLE AND POLT


personnel understand and follow closely the information
and guidelines for protection of clinical laboratory
workers issued by the Centers for Disease Control, other
federal agencies, and the NCCLS.
II.

M A T E R I A L S AND METHODS
This section is devoted to a general consideration of
safety equipment that is used to reduce the possibility of
transmission of infections to laboratory personnel
working with human and other tissues.
For any piece of safety equipment to be protective, it
must be used. If safety equipment is too complicated or
too uncomfortable, then the utilization of this equipment by laboratory personnel will be reduced. Thus,
with any safety equipment, one must compromise
between ease and comfort of use and its protective
characteristics. Another consideration may be cost.
A. Gloves.
Protective gloves vary considerably in their
strength, comfort, and in their interference with
dexterity of the hands. There is also a large variation
in the cost of protective gloves. The protective glove
should be matched to its use. Thus, for work which
requires a high degree of protection but less dexterity,
a very strong protective glove may be used such as
heavy duty (guage) latex or neoprene latex gloves.
Examples of such jobs are washing contaminated
dishes and instruments, performing autopsies, or
handling tissues when there is a reduced requirement
for manual dexterity. In other uses, such as in
dissection of organs or in tissue preparation, a high
degree of manual dexterity as well as a high degree of
protection may be required. In such cases more expensive, high quality, surgical gloves may be
necessary. Finally, in other uses that require a high
degree of manual dexterity but less risk of puncturing
or tearing the gloves, a glove that permits maximum
dexterity but does not require the great strength or
expense of surgical gloves might be used, such as
disposable latex gloves. Their advantage over surgical
gloves would be one of cost in that surgical gloves may
cost $1-$2 per pair, whereas 100 disposable latex
gloves can be purchased for approximately $15-$20.
An even less expensive glove, but one that offers less
dexterity, is polyethylene or polyvinyl chloride gloves.
All these gloves can be obtained from standard
scientific supply houses (e.g., American Scientific
Preducts) and the final selection of gloves depends on
the balance of ease of use, protection, strength, and
cost.
B. Face protection.
Protection of the face can range from safety glasses
to complete face masks. Contact lenses provide no
protection from splashes and may prevent adequate
cleansing following a splash to the eyes. Thus contact
lenses should never be worn in the laboratory unless
covered by goggles. As a minimum, safety glasses
should be used by all personnel requiring correction of
vision and goggles may be used by other personnel.
Safety glasses and goggles provide no protection for

192

mucous membranes of the nose and mouth. Also, eye


splashes can occur even when safety glasses which do
not have shields are being worn. To protect mucous
membranes, a total face shield can be used if the
situation requires such protection. Finally, where
aerosol exposure may be a problem, a total face mask
which can protect against fumes, aerosols, and particulate matter can be used. Such shields and goggles
can be obtained through safety catalogues {e.g., Lab
Safety Supply) or through the major scientific supply
houses.
C. Solutions for disinfecting (decontamination) of surfaces.
The solutions used for disinfecting {decontamination} of surfaces vary in their ability to kill or
destroy various organisms as well as their toxicity to
humans. Some agents disinfect well but are toxic to
man; other chemical agents are less toxic to man and
have intermediate activity in disinfecting surfaces
against various agents. Also, some chemicals are too
volatile {alcohols), and thus tile time the solution is in
contact with surfaces may be too short to ensure effective decontamination. These and similar factors are
considered when reaching a compromise concerning
the most effective agent to be used in decontaminating
laboratory surfaces. Household bleach ~Clorox)
currently is used as an effective viral decontamination
agent. The concentration used for decontamination
varies with the area to be cleaned because chlorine is
inactivated by protein. Thus if the surface is contaminated by blood, a higher concentration of chlorine
(e.g., full strength bleach) is necessary. For "clean"
surfaces potentially contaminated by infective agents a
1 to 10 dilution of bleach can be used. More resistant
agents, such as slow viruses, require full strength
bleach even for partial killing. The pros and cons of
various substances used to decontaminate infective
agents are discussed in detail. (18)
D. Surgical instruments.
Surgical instruments vary in quality and hence
price. In general, they require a high degree of care to
protect their mechanical characteristics. To disinfect
or clean surgical instruments consult the recommendations of the manfacturers of each instrument or
obtain guidelines through brochures prepared b y
surgical instrument manufacturers. For example, a
brochure prepared by Codman discusses the proper
way to clean and sterilize surgical instruments. In
general, surgical instruments should be cleaned in
distilled water and non-ionic detergent before
autoclaving. After initial cleaning, surgical instruments that have been in contact with human infective agents are autoclaved. Note: Autoclaving of
dirty instruments will accelerate corrosion and destroy
the characteristics of these expensive and sometimes
delicate tools.
If the research requires removing needles from
syringes, then a needle holder available from surgical
supply houses can be used to manipulate these needles.
Needles should never be removed from syringes by
hand.
Journal of TissueCulture Methods Vot.11, No. 4, 1988

GRIZZLE AND POLT

III.

PROCEDURE

A. Potential infectivity of tissues and body fluids from


humans
Most tissue sources do not distribute knowingly
tissues that are infected with dangerous pathogens.
However, all tissues, tissue products, and body fluids
from humans or primates must be considered as
potentially hazardous and must be handled as if they
were contaminated with the viruses causing AIDS or
hepatitis or with other pathogens. It is impossible to
exclude all infected cases because sources of the samples
must rely on historical information that cannot identify
asymptomatic infections. The following precautions
apply to most infective agents.
Most agents, including the virus that causes AIDS,
may remain infective in blood, body fluids, and tissues
even when they have dried 47}. Thus, great care should
be directed to laboratory cleanliness. The precautions
discussed below also apply to laboratory workers exposed to tissue cultures and to embryonated eggs,
animals, etc., that have been inoculated with tissue
products.
1. Serologic testing of patients
Patients are not tested routinely for antibodies to
HIV, hepatitis B, or other agents, so definite
identification of such infections is not possible. IT
IS ALSO USUALLY N O T POSSIBLE TO
DETERMINE
RETROSPECTIVELY
W H E T H E R SUCH I N F E C T I O N S E X I S T E D
IN P A T I E N T S FROM WHOM T H E TISSUE
WAS O B T A I N E D .
B. Good general laboratory practices --summary
1. Always wear gloves when handling tissues or body
fluids.
2. Always wear a closed-front coat, gown, or uniform
in the laboratory.
3. Do not smoke, eat, drink, or apply cosmetics in the
laboratory.
4. Do not handle clean surfaces, material or equipment, or touch yourself or others with contaminated gloves.
5. Use mechanical pipetting devices; never pipette by
mouth.
6. Avoid using syringes with needles to transfer
potentially infectious liquids.
7. Avoid procedures that may cause aerosols or
droplets to form; use containment for all such
procedures.
8. Decontaminate all work surfaces with sodium
hypochlorite solution (1:10 solution of household
bleach} following completion of work and with
undiluted bleach when cleaning up spills.
9. Decontaminate all potentially contaminated
material before disposal.
10. Discard gloves in appropriate container, remove
protective laboratory clothing, and wash hands
before leaving the laboratory.
11. Do not allow contaminated glassware, equipment,
and instruments to accumulate in the laboratory.
Clean them as soon as practicable after use.
Journal of Tissue CultureMethods Vol.11, No. 4, 1988

C. Good laboratory practices--general laboratory safety


precautions
The two important safety precautions in the
laboratory are to wear gloves consistently and correctly
and to wash hands frequently. The use of gloves can not
be considered a substitute for careful hand washing after
working with potentially infectious material.
1. Hand washing
Hands are to be washed thoroughly using an
effective detergent. It is a good practice to wash
hands after each batch of analytical tests. Always
wash hands before and after eating, drinking,
smoking, or using the bathroom, and after completing your work. Do not handle the telephone,
door knobs, or laboratory equipment with contaminated hands or gloves. Always wash hands
after contact with patients, animals, or specimens
and before leaving the laboratory.

2. Gloves
a. Wear gloves whenever there is any possibility of
contamination {e.g., handling specimens, test
vessels, or containers), particularly if hands are
cut or abraded. Gloves may become contaminated while handling specimens and
reagents and must not then be used to handle
clean objects te.g., laboratory equipment,
doors, records, etc.}. Discard gloves (into
contaminated waste) immediately after use to
minimize the possibility of contamination.
i. Wear disposable gloves when handling tissue
specimens, when removing plasma or serum
from blood, and when handling any human
fluids or performing any tests that use human
fluids.
ii. Wear gloves while changing dialyzer membranes on automated chemistry equipment or
any other cleaning operation on equipment
when hands are likely to contact residual
human biological specimens or contamination directly.
iii. Do not touch unprotected areas of the body
{e.g., eyes, face, mouth, etc.) while wearing
gloves.
iv. Remove and properly dispose of gloves before
leaving the work area and before touching
telephones, procedures, reports, logs,
doorknobs, etc.
v. If in ususual circumstances it becomes
necessary to touch the telephone, doorknobs,
or other clean objects while wearing gloves,
use a paper towel (discard appropriately) to
handle the equipment and disinfect equipment immediately after use.
vi. Any puncture or tear of the glove renders the
glove useless. Discard the glove and replace
it immediately.
vii. Use disposable gloves only once and
decontaminate reusable gloves after use.

Always wash hands thoroughly after removing gloves.


193

GRIZZLE AND POLT


3. Laboratory clothing
Wear gowns, laboratory coats, uniforms, or
other protective clothing when working. Protective
clothing should be kept closed le.g., buttoned,
zipped, snapped, etc.} to minimize contamination
of personal clothing. This permits easy removal and
appropriate storage or disposal of contaminated
clothing before leaving the laboratory area.
Protective clothing should be decontaminated
following any contact with human blood, tissue, or
fluids. An appropriate periodic decontamination
schedule te.g., weekly) should be established for
protective garments even if no obvious contamination has occurred. The autoclave is the most
reliable method of decontaminating laboratory
clothing. Avoid garments with a high percentage of
acetate or other highly flammable material.
4. Eye, nose, mouth protection
Face shields, eye protectors, eye glasses and
other protective barriers should be used when
handling infectious agents, caustic materials, or
liquid nitrogen. Inasmuch as the mucous membranes of the eyes have been identified as a transmission route for some viral infections, goggles or
face shields may be needed for some preparative
work. Contact lenses provide no protection and
may hinder decontamination in some cases.
Workers should be encouraged to wear safety
glasses with splash shields when other protection is
not deemed necessary. Whenever there is any
possibility of splashes or of aerosols, wear face
shields or take other protective measures to prevent
exposure of mucous membranes of the eyes, nose,
and mouth.
5. Smoking, eating, drinking, cosmetics, and contact
lenses
Smoking, eating, drinking, and applying
cosmetics can be sources of contamination to
workers, specimens, laboratory equipment, and
reagents. Laboratory personnel can be exposed to
infectious agents in blood, tissues, and body fluids
via either the oral or parenteral route. Specimens
stored in laboratory refrigerators may contain a
variety of pathogens. For these reasons, there
should be no smoking, drinking, or eating within
the laboratories. Also, items for human consumption (e.g., food, ice, water) are not to be stored
in laboratory refrigerators. Fingers, pencils, and
other items that may become contaminated must be
kept away from the mouth, nose, and eyes. No
foods are to be permitted in the laboratories or
other areas in which specimens are collected and
handled. Coffee makers must not be allowed in
areas where specimens are collected, processed, or
handled. Eating, drinking, and smoking may be
permitted in clean areas such as offices, conference
rooms, and corridors. Cosmetics or contact lenses
must not be applied in the laboratory. Their application constitutes a potential hazard because
infectious agents can be inoculated thereby into the
mouth or eyes.
194

6. Prevention of direct contamination


a. Sharp items (needles, scalpel blades, and other
instruments} must be considered potentially
infective and should be handled with extraordinary care to prevent accidental injuries.
b. Broken or chipped glassware must be discarded
or repaired. Consider all glassware that has been
exposed to tissues, tissue products, or body
fluids to be contaminated.
c. Substitute unbreakable, e.g., plastic, items
whenever possible.
d. Do not pick up broken pieces of glass or glassware
with bare hands. Broken glassware must be
disposeed of in puncture-resistant containers
and preferrably autoclaved before disposal if
there is any possibility the glassware is contaminated.
e. Do not attempt to remove rubber stoppers or glass
tubing by force; cut them off if they are stuck.
f. Use hypodermic needles and syringes only for
parenteral injection and aspiration of fluids
from laboratory animals and diaphragm bottles.
Use only needle-locking syringes or disposable
syringe-needle units (i.e. needle is integral to the
syringe) for the injection or aspiration of infectious fluids.
g. Replace sharp needles with blunt needles or
cannules whenever possible.
h. Use extreme caution when handling needles and
syringes to avoid autoinoculation and generation
of aerosols during use and disposal.
i. To prevent needlestick injuries, do not recap
needles nor purposely bend, break, remove from
disposable syringes, or otherwise manipulate
needles by hand.
j. Place disposable syringes, needles, scalpel blades,
and other sharp items in puncture-resistant
containers located as close as practicable to the
area in which they are used. They should be
decontaminated, preferably by autoclaving,
before being discarded.
7. Aerosols
a. Aerosols generated from biological specimens are
a major potential source of infection. Perform
atl procedures and manipulations of potentially
infectious material carefully to minimize the
creation of droplets and aerosols.
b. Biologic safety cabinets (class I or II} and other
primary containment devices (e.g., centrifuge
safety cups} are advised for all procedures that
are likely to create aerosols or infectious
droplets. These procedures include centrifuging,
blending, sonicating, vigorous mixing, and
harvesting infected tissues from animals or
embryonated eggs.
c. Droplets generated by fluorescent-activated cell
sorters represent potentially infectious aerosols.
Translucent plastic shielding between the
droplet-collecting area and the equipment
operator may be used to reduce the presently
uncertain magnitude of this risk.
Journal of Tissue Culture Methods

Vol. 11, No. 4, 1938

GRIZZLE AND POLT


d. Use primary containment devices when handling
materials that might contain concentrated infectious agents or organisms in quantities
greater than those expected in clinical
specimens.
e. Blood specimens are normally collected in a
vacuum-type tube. Frequently, some vacuum
remains after the blood is drawn and thus it is
difficult to remove rubber stopper. "Popping"
the cork can generate droplets or an aerosol that
could conceivably cause infection or may cause
exposure due to splashes or both. Laboratory
personnel should twist the cork gently, first
covering it with absorbent paper or cloth (e.g.,
Kim Wipes} to minimize aerosols. Dispose of
protective papers into proper containers for
contaminated waste.
f. If a fluid specimen must be emptied from a
syringe, CAREFULLY, using a needle holder
or other instrument ~NOT T H E HANDSL
remove the needle to minimize aerosols. Insert
the syringe deep into a large container and allow
the fluid to run down the inside of the container.
8. Opening, pouring and spilling of biological
specimens
a. Removing the stopper from a tube may produce
hazards other than aerosol production ~see
aerosol precautionsL such as possible contamination of the outside surface of the tube.
Contamination may occur when removing the
stopper, while filling the tube, or through
leakage.
b. Should blood or other biological material contaminate the outside, disinfect the tube and wipe
clean with an appropriate germicidal agent
before the tube is stored or handled by anyone
else.
c. The hazard of contaminated specimen containers
may be reduced if liquid specimens are transferred using a bulb suction pipette or other
mechanical device rather than by .pouring.
d. Clean blood spills promptly with a disinfectant
solution, such as sodium hypochlorite (undiluted household bleach).
e. Other infections such as cryptosporidium and
myobacteria may occur concurrently in AIDS
patients; therefore, additional precautions may
be required because clinical specimens may
contain other infective agents.
9. Pipetting
a. Pipetting of all reagents and specimens is always
performed mechanically using a rubber bulb or
other safety device, never by mouth. This includes test materials, diluents, reagents, and
other noninfectious materials as well as infectious materials. Not only is there danger of
aspirating infectious material, but mouth pieces
of the pipettes may become contaminated by the
worker's hands and by aerosols. Thus, infectious agents can be ingested even through
mouth pipetting of noninfectious material.
Journal of Tissue CultureMethods Vol. 11, No. 4, 1988

Contaminated pipetting devices can be


decontaminated using a detergent solution
containing an effective concentration of germicide. When capillary tubes are used for
dispensing serum, decontaminate the small
rubber bulb after each test run by disinfecting
for at least 10 min. Note: capillary tubes may be
easily broken and, if broken, pose a hazard,
especially when they have been exposed to
human material.
b. After use, lay pipettes lincluding re-usable
Pasteur pipettes) flat in a container of disinfectant solution. Do not drop pipettes into a
vertical pipette holder as this is likely to produce
aerosols due to fluid rapidly rising in their
lumens.
c. Do not leave pipettes sticking out of bottles,
flasks, or beakers as this provides a potential
source of injury and contamination.
10. Centrifugation
a. Clean centrifuges and serofuges daily using an
appropriate gemicidal solution such as 0.525%
sodium hypochlorite tl:10 dilution household
bleachL They should also be cleaned after each
run involving known contaminated specimens.
b. Aerosol production daring centri/agation can
contribute to the spread of viruses. Cover
centrifuge tubes whenever feasible, preferably
with tight-fitting caps. Laboratory centrifuges
should be located to the extent practicable away
from laboratory personnel and if possible in
areas or rooms with inward airflow.
c. Close the top of the centrifuge while the centrifuge
is operating. Do not open until the unit has fully
stopped.
d. Orientate serofuges of the type used in blood
banks so that the air exhausting from the vent
located at the base of the centrifuge is directed
away from the operator. This is recommended
to minimize aerosol inhalation.
e. Instruct each centrifuge operator on proper
operation procedures before he/she is allowed to
use the centrifuge. Instructions must include
balancing loads, using the proper rotor, and
using accessory equipment. Each employee who
uses a centrifuge is responsible for the condition
of the machine at the end of the procedure. This
includes entering data in the log book, turning
off the power, cleaning up spills and broken
glass, decontamination, etc.
f. If the loads in centrifuges are unbalanced,
biological fluids may splash from the centrifuge.
Inasmuch as rotor defects or breakage may
cause unbalancing at high speeds, check the
condition of the rotor periodically.
g. Centrifuge tubes used in angle-head centrifuges
must never be filled to the point that liquid
contacts the lip of the tube. Although the
meniscus will be vertical during rotation, high
forces can drive the liquid past the cap seal and
over the outside of the tube.
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GRIZZLE AND POLT


h. Clean the centrifuge thoroughly with appropriate
cleaning agents after breakage or contamination. Disinfect carrier cups whenever a
sample is spilled.
11. Specimen containers and transport
a. Label blood and other specimens from humans
prominently with a special warning that the tube
contains potentially infective material. Label
tubes before they are used.
b. Clean specimen containers that are visibly
contaminated with blood with a disinfectant
such as 5.25% sodium hypochlorite tundiluted
household bleach).
c. Place blood specimens in a second container, such
as an impervious bag, for transport. Examine
the containers or bags carefully for leaks or
cracks before transport.
12. Disposable items and specimens
a. Extreme care must be taken when disposing of
any item that contains or has contained
biological specimens.

b. Place the items listed below in containers


designed for biologically contaminated material.
When scheduled for disposal, the containers are
closed and placed in boxes marked "Contamination" for incineration or other appropriate disposal. Separate glass and other
noncombustibles from items that can be incinerated easily.
Pipettes
Sputum
Capillary tubes
Stool
Specimen tubes
Bronchial fluid
Culture plates
Spinal fluid
Other glassware
Gastric washings
c. Special precautions are required for the items
listed below:
i. Needles. Place in a strong box marked for
disposal of contaminated needles and blades.
if. Syringes with needles. Place in trash container
appropriate for and marked for disposal of
contaminated glassware or in box marked for
disposal of needles.
iii. Surgical blades. Place in box with contaminated needles.
iv. Sample containers. Non-glass: Place in a
waste container marked for incineration.
Glass: Place in a container for disposal of
contaminated glassware.
v. Tissues, blood, or serum. Place in a waste
container marked for incineration or dispose
in accordance with other applicable institutional safety procedures.
d. Take care not to splash fluids on walls, countertops, floor, or equipment when placing
sample cups and tubes into the waste container.
Clean any spills immediately with an effective
decontaminating solution.
13. Reusable items
The following are routine laboratory techniques
for reusable items:
196

a. Soak syringes, needles, pipettes, glassware, and


specimen tubes at least 10 min in an appropriate
disinfectant or detergent or autoclave after
cleaning.
b. Soak rubber bulbs at least 10 min in disinfectant
detergent.
Note: Cleaning with some detergents such as
Duponot may not destroy gram negative bacteria
and fungal agents.
14. Laboratory records
Laboratory records can serve as a source of
infection when contaminated with blood, serum, or
urine. Be extremely careful to avoid such contamination. Laboratory records must be considered
contaminated after any contact with biological
material, and hepatitis may be transmitted via cuts
by contaminated paper ~20). Laboratory records
are never carried from laboratory areas into clean
areas. Thus, it is good general practice to keep
requisitions, experimental records, and other
paperwork away to the extent practicable from
specimens to minimize contamination.
15. Long hair and beards
Secure hair and beards to prevent their contact
with contaminated materials or surfaces. This will
minimize transfer of organisms to or from the work
area and thereby diminish opportunities for infection. This is also a good safety precaution
around moving equipment.
16. Sterilization, disinfection, housekeeping, and waste
disposal to prevent transmission of viral infection
a. The infectivity of some viral agents may be
reduced or abolished by heat. For example, HIV
infectivity is abolished by heating samples at 56
C for 30 min (21). Some proteins, other biologic
activities, and measurements are unaffected by
such treatment (12). Thus, heat inactivation of
samples may be useful in the protection of
laboratory workers from H I V / o r certain other
viral agents or both.
b. Sterilization and disinfection procedures
currently recommended for use in health care
and dental facilities are adequate to sterilize or
disinfect instruments, devices, or other items
contaminated with the blood or other b o d y
fluids from persons infected with HIV or
hepatitis.
c. Surgical instruments or other nondisposable items
used in all procedures are decontaminated after
use rather than just rinsed with water.
Decontamination can be done by machine or by
hand cleaning by trained personnel wearing
appropriate protective attire, including gloves,
and using appropriate chemical germicides.
This can be followed by autoclaving to achieve
high-level decontamination.
d. Decontamination is required for instruments or
other nondisposable items that touch intact
mucous membranes.
e. Several liquid chemical germicides commonly
used in laboratories and health care facilities
Journal of Tissue CultureMethods Vol.11, No. 4, 1988

GRIZZLE AND POLT


have been shown to kill HIV and hepatitis
viruses at concentrations lower than those used
in practice (18).
f. Chemical germicides registered with and approved by the Environmental Protection Agency
(EPA) as "sterilants" can be used either for
sterilization or for high-level decontamination of
instruments or medical devices, depending on
contact time (18).
g. Germicides that are approved as "hospital
disinfectants" and are mycobactericidal in
appropriate dilutions can also be used for highlevel decontamination of devices and instruments.
h. Germicides that are mycobactericidal are
preferred because mycobacteria represent one of
the most resistant groups of microorganisms;
therefore, germicides that are effective against
mycobacteria are also effective against other
bacterial and viral pathogens except slow viral
agents (see Creutzfeldt-Jakob discussion).
i. When chemical germicides are used, instruments
or devices to be sterilized or disinfected are to be
thoroughly cleaned before exposure to the
germicide, and the manufacturer's instructions
for use of the germicide are to be followed.
j. Hospitals generally use adequate laundry and
dishwashing cycles to decontaminate linens,
dishes, glassware, and utensils; however, grossly
contaminated items should be decontaminated
before washing.
k. Housekeeping procedures commonly used in
hospitals are usually adequate for cleaning
environmental surfaces. Clean surfaces exposed
to blood and body fluids are cleaned with a
detergent followed by decontamination using an
EPA-approved hospital disinfectant that is
mycobactericidal. Personnel should wear
protective gloves and garments in cleaning up
spills and during cleaning procedures in general.
]. Decontaminate laboratory work surfaces with a
disinfectant after any spill of potentially infectious material and also upon the completion
of work activites. Decontaminate spills with
undiluted bleach, and other surfaces with a 1:10
dilution of bleach. For large, relatively clean
surfaces, a dilution of 1:100 is adequate for most
infective agents, except for the rare CreutzfeldtJakob agent, which requires undiluted bleach
(18).
m. Remember individuals who are unfamiliar with
the fact that surfaces are contaminated may be
exposed to these surfaces when your workplace
is unattended.
n. Sharp items are considered as potentially infective
and are handled and disposed of with extraordinary care to prevent accidental injuries.
o. Other potentially infective wastes are contained
and transported in clearly identified, impervious
plastic bags. If the outside of the bag is contaminated with blood or other body fluids, a
Journal of Tissue Cukure Methods

Vol. 11, No. 4, 1988

second outer bag is used. The bags are not to be


loaded to the breaking point.
p. Recommended practices for diposal of infective
waste are adequate for disposal of waste contaminated by HIV. If necessary, blood and
other body fluids may be poured carefully down
a drain connected to a sanitary sewer; however,
this should be followed by extensive flushing,
preferably with undiluted bleach. If a drain used
to dispose of human wastes requires repair,
inform personnel performing the work that the
drain is contaminated so that these personnel
can take adequate precautions.
q. Decontaminate all potentially contaminated
materials used in laboratory testing, preferably
by autoclaving, before disposal or reprocessing.
Do this daily to minimize unintentional exposure to non-laboratory personnel.
r. Institutions that are not associated with medical
centers may obtain guidance in selection of
sterilization procedures and agents from the
infection control committees of local hospitals
and from reference 18.
D. Hepatitis
The medical literature contains a large body of data
indicating that hospital personnel are significantly at
risk for viral hepatitis (10,11 }. Researchers using human
tissues and body fluids are also at risk. Recent reports
have substantiated this, and further indicate that
personnel in the clinical laboratory of the hospital are at
greater risk than many other hospital employees.
1. Reasons for the high incidence of hepatitis among
clinical laboratory workers
a. Frequent close personal contact by some personnel with patients with hepatitis.
b. Direct and frequent contact with biological
specimens containing the viruses.
c. Frequent opportunity for accidental puncture
wounds with contaminated needles and sharp
objects.
d. Carelessness in handling specimens, in wearing
gloves, and in using proper and frequent handwashing technique.
e. Inadequate or unsafe disposal of contaminated
needles, specimens, or other objects.
2. Control sera and biological reagents
a. Commerical sera may contain the hepatitis antigen or other viruses. Conscientious suppliers
will usually indicate whether the specimen has
been tested for HB-Ag or HIV or both.
b. Up to 60% of the typing and control sera contain demonstrable HB-Ag and should be
considered as major sources of infections
hepatitis virus and possibly of HIV.
c. HB-Ag positive commercial controls have been
found in blood bank, coagulation, hematology,
chemistry, and serology reagents.
d. Control sera or other reagents must not be mouthpipetted and must be treated with the same
degree o f caution as patient specimens.
e. Infective agents may be transmitted to laboratory
197

GRIZZLE AND POLT


workers through puncture wounds, skin
abrasions, eyes and nose Isplashing materialL
cuts or skin lesions Ipaper cuts e.g., data sheetst,
aerosols linhaled into the respiratory tractL
mouth (oral routeL and direct contact with
patients.
f. Because transmission may occur via any number
of unsuspected routes, laboratories are to
develop detailed techniques and to train personnel in their careful application to minimize
exposures to viral pathogens.

g. Safe laboratory practice is to be followed closely


and applies to aH individuals working or visiting
the laboratory work areas, regardless of title,
position, day of the week, or hour of the night or
day.
E. AIDS
Human immunodeficiency virus (HIV) talso known
as human T-cell lymphotrophic virus-III [HTLV III]
and lymphadenopathy associated virus [LAV]) is
transmitted primarily through homosexual and
heterosexual contact; parenteral exposure to infected
blood or blood components, e.g., contaminated needle
sticks; and perinatal transmission from mother
to neonate. HIV has been isolated from blood,
cerebrospinal fluid, semen, saliva, tears, breast milk,
vaginal secretions, and urine, and is likely to be isolated
from other body fluids, tissues, secretions, and excretions. Epidemiologic evidence, however, has implicated primarily blood, vaginal secretions, and semen
in transmission. There is some evidence that in very rare
individuals, a minute exposure to the HIV virus via
needle sticks or blood splashes may result in transmission of HIV. Inasmuch as HIV and hepatitis B
have become common infections of hospitalized
patients, all tissues, tissue products, and body fluids
from humans and primates must be considered
potentially infective.
1. Precautions to prevent acquisition of HIV infection
by research workers
a. Studies indicate that viral hepatitis is more readily
transmitted via human tissue and tissue
products than is HIV (11). Thus, precautions
effective for viral hepatitis represent prudent
practices that apply to preventing transmission
of HIV and most other blood-borne infections.
These guidelines have been discussed
previously.
b. The risk of transmission of HIV to research
workers seems to be low and can be further
minimized when routinely recommended infection-control precautions are followed
(10,11,18L
c. Serologic testing should be available to workers
who may wish to know whether they have been
infected previously with HIV or viral hepatitis.
d. Workers who have been exposed to potentially
infected material should be followed per
198

governmental guidelines to evaluate whether


they have been infected.
e. Recent data indicate that current antibody" tests
may not show positive results for as long as 2 yr
after exposure. Workers who have been
potentially infected should be informed of this
and appropriate follow-up recommended ( 7L
2. Risk of occupational acquisition of other infectious
diseases by health care workers infected with HIV
or other immunosuppressive conditions
a. Health care workers who are known to be infected
with HIV and/or who have compromised
immune systems from other etiologies (e.g.,
steroid therapy} are at an increased risk of
acquiring or experiencing serious complications
of other infectious diseases. Of particular
concern is the risk of severe infection after
exposure to patients with infectious diseases,
e.g., tuberculosis, that are easily transmitted if
appropriate precautions are not taken.
b. Counsel all workers infected with HIV or who
have compromised immune systems secondary
to other conditions concerning the potential risk
associated with human tissues and fluids.
c. The worker's personal physician, in conjunction
with the institution's employee health services,
determines on an individual basis whether a
compromised worker can perform research
duties adequately and safely and recommends
changes in work assignments, when indicated.
d. These immune-deficient workers who continue in
the laboratory should follow carefully existing
recommendations for infection control to
minimize their risk of exposure to other infectious agents.
e. Pregnant research workers are not known to be at
greater risk of contracting HIV infections than
health care workers who are not pregnant.
However, workers who develop HIV, hepatitis,
or other viral, bacterial, or amoebic infections
during pregnancy risk fetal infection through
perinatal transmission. Pregnant research
workers especially should foUow careful
laboratory practices.
F. Creutzfeldt-Jakob agent precautions
The Creutzfeldt-Jakob agent is responsible for the
transmission of certain dementias in man. Fortunately,
this agent is extremely rare in the population of the
United States. It represents an extra danger, however,
because it remains infectious even after treatment with
most detergents, formalin, tissue processing to paraffin
blocks, or agents that degrade DNA and R N A tl8L
This agent can b e inactivated by phenol, by 5.0%
sodium hypochlorite, and by autoclaving at 121 C and
20 psi for 60 min tl8L Some researchers believe similar
agents may remain infective even after the above
treatments.
G. New developments in infection control
With increased attention to transmission of viruses via
blood products, new viral agents associated with
diseases have been identified in the blood supply. These
Journal of Tissue CultureMethods Vol.11, No. 4, 1988

GRIZZLE AND POLT

include human T-cell lymphotrophic virus-I tHTLV-I)


that has been associated with T-cell malignancies. The
Center for Disease Control (CDC) frequently updates
information concerning HIV, hepatitis, and other infective agents and disseminates this information via the
Morbidity and Mortality Weekly Report ~MMWR).
This report should aid investigators in closely following
new developments in infection control. Also, the
NCCLS has prepared a preliminary draft for clinical
laboratories of proposed guidelines to minimize transmission of infective agents {19). These guidelines
provide detailed information beyond the scope of this
article. Similarly a very important guide to laboratory
safety and the prevention of intralaboratory infections is
the book by Miller (18).

V.

REFERENCES

1. Centers for Disease Control, Acquired immune deficiency syndrome


tA1DS): precautions for clinical and laboratory staffs. Morbidity and
Mortality Weekly Report ~MMWR) 31:577-580; 1982.
2. Centers for Disease Control. Acquired immunodeficiency syndrome
iAIDS): precautions for health-care workers and allied professionals,
MMWR 32:450-451; 1983.
3. Centers for Disease Control. Recommendations for preventing possible
transmission of human T-lympbotropic virus type III/lymphadenopathy-associated virus from tears. MMWR 34:533-534; 1985.
4. Centers for Disease Control. Recommendations for preventing transmission
of infection with HTLV-III/LAV in the workplace. MMWR
34:682-695; 1985.
5. Centers for Disease Control. AIDS recommendations and guidelines.
November 1982-November 1986.
6. Centers. for Disease Control. Revision of the CDC surveillance case
definition for acquired immunodefieiency syndrome. M~IWR Supplement 1S, 36:ls-15s; 1987.

7. Centers for Disease Control. Recommendations for prevention of HIV


transmission in health-care settings. MMWR Supplement 2S,
36:ls-18s: lq~7 R-r,-;~ted in Lab. Med. 19:88-95; 1988.
8. College of American Pathologists. Summary of recent information:
acquired immunodeficiency syndrome. July; 1983.
9. Conte, J, E,, Jr.; Hadley, W. K.; Sande, M., et al, Infection-control
guidelines for patients with the acquired immunodeficiency syndrome
tAIDSL N. Engl. J. Med. 309:740-744; 1983.
10. Favero, M. S. Biological hazards in the laboratory. Lab. Med. 18:665-670;
1987.
11. Gerberding, J. L.; Bryant-LeBlanc, C. E.; Nelson, K., et al. Risk of
transmitting the human immunodeficiency virus, cytomegalovirus, and
hepatitic B virus to health care workers exposed to patients with AIDS
and AIDS-related conditions. J, Infect. Dis. 156:1-8; 1987.
12. Goldie, D. J.; McConnell, A A.; Cooke, P. R. Heat treatment of whole
blood and serum before chemical analysis. Lancet 1161 :May; 1985.
13. Haber, S. L. Part h What every laboratorian should know about AIDS.
Med. Lab. Observer November: 32-38; t985.
14. Haber, S. L. Part 2: What every laboratorian should know about AIDS.
Med. Lab. Observer December:55-59; 1985.
15. HHS Publication No. tCDC) 84-8395. Biosafety in microbiological and
biomedical laboratories. 1st cd. March; 1984.
16. Maas, A. E. AIDS autopsy precautions. Pathologist November:20-21;
t 985.
17. MacArthur, S.; Schneiderman, H. Infection control and the autopsy of
persons with human immunodeficiency virus. Am. J. Infect. Control
15:172-177; t987.
18. Miller, B. M,, ed, Laboratory safety: principles and practices. Washington,
D.C.: American Society for Microbiology; 1986.
19. National Committee for Clinical Laboratory Standards (NCCLS} Proposed
Guidelines. Protection of laboratory workers from infectious disease
transmitted by blood and tissue. NCCLS Document M29-P, vol. 7 no.
9; 1987.
20. Pattison, C. P.; Boyer, K. M.; Maynard, J. E., etat. Epidemic hepatitis in
a clinical laboratory: possible association with computer card handling.
JAMA 230:854-857; 1970.
21. Spire, B.; Dormont, D.; Barre-Sinoussi, F., et al. Inactivation of lymphadenopathy-associated virus by heat, gamma rays, and ultraviolet
light. Lancet. January:188-189; 1985.
22. Valenti, W. M. AIDS and the lab: infection control guidelines. Med. Lab.
Observer February:53-56; 1986.
23. Wofsy, C.; Kaplan. L.; Volberding, P. Clinical and laboratory features of
HIV infection. Abbott Park, IL: Abbott Diagnostics Educational
Services. September; 1987.

Supported by National Institutes of Health Grant CA 44968.

Journal of Tissue Culture Methods

Vol. 11, No. 4, 1988

199