Safety Precautions for Performing Tests for
Hepatitis-associated "Australia"
Antigen and Antibodies
NATHALIE J. SCHMIDT, P H . D . , AND EDWIN H. LENNETTE, M.D.,
PH.D.
Viral and Rickettsial Disease Laboratory, California State Department of Public Health,
Berkeley, California 94704
ABSTRACT
Schmidt, Nathalie J., and Lennette, Edwin H.: Safety precautions for performing tests for hepatitis-associated "Australia" antigen and antibodies.
Am. J. Clin. Pathol. 57: 526-530, 1972. T h e hazards of infection involved
in performing tests for hepatitis-associated "Australia" antigen are discussed.
General microbiologic safety procedures which should be employed in handling potentially infectious blood and blood products are indicated. Specific
measures which may be taken to reduce the risk of infection in conducting
immunodiffusion, immunoelectroosmophoresis, and complement fixation tests
are presented.
BLOOD and blood products containing the so-called "Australia" or hepatitis-associated antigen (HAA) have been
shown to produce the "serum," or long
incubation, type of hepatitis in a high
proportion of recipients.1"3-7 It has not
been determined whether the antigen is
actually the hepatitis virus or a subunit of
the infectious agent, but the infectivity of
blood specimens containing the antigen
has been clearly established. Assays for
HAA are now being conducted in many
blood banks as a means of screening to
detect those blood specimens and blood
products which might produce hepatitis.
The handling and testing of specimens
containing HAA is potentially hazardous,
and some of the technical personnel performing the tests may not be adequately
trained in microbiologic procedures required for safe handling of infectious materials. Certain precautionary measures have
been adopted in this laboratory in an ef-
HUMAN
Received March 1, 1971; manuscript resubmitted
June 28, 1971; accepted for publication July 12,
1971.
fort to minimize the hazards involved in
testing blood specimens which may contain hepatitis viruses, and these are described herein as a possible aid to laboratory workers who may lack background
and experience in the technics necessary
for the safe handling of infectious agents.
General Safety Precautions
It is now established that both the
"serum" and "infectious" forms of hepatitis can be transmitted by infectious blood
through either the oral or parenteral route. 4
Therefore, there should be no smoking,
eating, or storage of food in laboratories
where blood specimens are handled and
tests performed. The infectious agent(s)
may be transferred onto cigarettes or food
by contaminated hands of the laboratory
worker or through aerosols. Cases of hepatitis with "Australia" antigenemia are reported to have occurred in hospital and
laboratory personnel for whom a possible
source of exposure was food stored in the
same refrigerator with blood specimens.5
526
April 1972
SAFETY PRECAUTIONS FOR HAA TESTS
Pipetting of all reagents should be done
using a rubber bulb or other safety device
(but not a mouthpiece). This applies to
the test materials and to diluents and
other noninfectious reagents as well. Not
only is there a danger of aspirating infectious material through mouth pipetting,
but pipette mouthpieces become contaminated by the worker's hands and by aerosols, and thus infectious agents can be ingested even through mouth pipetting of
noninfectious materials. Mouthpieces of pipettes should be cotton-plugged to prevent
contamination of the attached bulb. If capillary tubes are used for dispensing test reagents, the small rubber bulbs should be
decontaminated after each test run by autoclaving or by boiling them for 30 min.
All tests should be performed on a working surface which can be decontaminated
effectively. In this laboratory the benches
are covered with plastic-backed paper*
which is impervious to fluids. Any accidentally spilled material can easily be seen
on the paper and avoided, and it is prevented from soaking through to the bench
top by the plastic backing. The paper is
discarded and autoclaved after completion
of the test run.
Only the materials needed for the immediate test procedure should be in the
working area; books, records and other
items which cannot be properly decontaminated should be kept away from exposure
to possible contamination.
In addition to items which have been
in direct contact with infectious materials,
all of the other glassware, instruments,
leftover diluents, etc., which have been in
the working area during the conduct of the
test, and thus have been exposed to possible contamination, should be placed directly into metal discard pans and decontaminated by autoclaving. Tests to be
• Crown Poly paper, hospital roll, towel stock,
bleached white with extruded polyethylene backing
blended into paper fibers. Union Paper Company.
527
discarded, e.g., slides or plates from immunodiffusion or immunoelectrophoresis tests
and plates or tubes from complement fixation tests, are placed in metal discard pans
for autoclaving; the contents are not emptied from the test vessels prior to decontaminating, as this may produce aerosols
of infectious material.
Wherever feasible, rubber or plastic disposable gloves may be worn for handling
specimens and test vessels or containers;
this is particularly important if there are
cuts or abrasions on the hands. However,
it should be recognized that gloves become
contaminated through handling specimens
and reagents, and a glove which has been
used for holding infectious materials should
not then be used for handling laboratory
equipment, records, etc. Instead, immediately after use the gloves should be
placed in a metal discard pan for decontamination by autoclaving or boiling. The
use of gloves should not be considered a
substitute for careful hand-washing after
working with infectious materials.
Extreme care should be taken in handling syringes and needles to prevent accidental inoculation with serum or blood
which might contain hepatitis viruses.
From the standpoint of potential infectivity, serum or plasma is stored more
safely in screw-capped vials than in tubes
with rubber or cork stoppers. Removing
the stopper from a tube is likely to produce
an aerosol of infectious material, particularly if the stopper has become moistened
by the contents of the tube. Also, with stoppered tubes, possible contamination of the
lip which may occur during filling or
through leakage presents a greater hazard
to the worker. The mouth of a tube or
vial containing serum or plasma should be
flamed after opening and before replacing
the closure to inactivate infectious agents
which may have been transferred to the
lip of the container.
528
SCHMIDT AND LENNETTE
Precautions for Individual Test Procedures
Immunodiffusion Tests. Some of the immunodiffusion technics described for assay
of HAA utilize a gel on a flat glass slide.
It should be recognized that the surface of
the gel becomes contaminated in the course
of filling the wells, and further, infectious
agents in the sera may diffuse to the surface
of the gel and to the edges of the slide.
During incubation of the test in a humid
atmosphere, moisture forms on the gel and
slide, and this may serve to contaminate
the edges and back of the slide and the
container in which it is held. This creates
a hazard in holding the slide for reading
the reactions. The use of a gel contained
in a petri dish or plastic cup is safer, as it
minimizes the possibility of contamination.
In this laboratory, immunodiffusion tests
for hepatitis-associated antigen and antibody are conducted in flat-bottomed cups
in a plastic panel.t 8 A section containing
the number of cups needed for the test
run is cut from a large panel. Melted agarose solution is added to each cup in a
volume of 0.5 ml. and allowed to solidify.
After wells are cut in the agarose gel, the
panel section is placed on top of a moistened filter paper disk in a large petri dish,
and the reagents are added to the appropriate wells using capillary pipettes. The lid is
then placed on the petri dish and sealed
with masking tape for incubation of the
test. Forceps with bent blades are used for
removing the panel of cups from the petri
dish and for manipulating the cups over
the reading light. The forceps are autoclaved after use.
Staining the immunoprecipitates is a potentially hazardous procedure, and it is
questionable whether the sensitivity of the
test is increased sufficiently to warrant the
risk involved. The rinse fluids and staining
solutions become contaminated with infectious material from the gel, as does the
f Model FB-54, Linbro Chemical Company, New
Haven, Conn.
A.J.C.P.—Vol. 57
blotter paper used for drying the gel. It
is likely that infectious material is distributed over the entire slide in the staining
process, and this makes later handling,
reading, and storage of the slides potentially dangerous. It has been found in this
laboratory that stained slides may be subjected to ethylene oxide sterilization without destroying or distorting the immunoprecipitates, and this can be performed on
stained slides which are to be stored. In
our experience, careful reading of immunodiffusion tests for HAA against a black
background using oblique light detects as
many positive reactions as are detected by
staining the immunoprecipitates.
Immunoelectroosmophoresis
(IEOP)
Tests. The hazards of contamination inherent in performing immunodiffusion
tests on flat glass slides also apply in the
case of IEOP tests, as these are performed
in a gel on a glass plate or on a cellulose
acetate membrane. In addition to the test
plate, the wicks conducting the buffer from
the electrophoresis chamber to the plate,
the buffer, and the chamber itself may all
become contaminated during the run.
After the wells have been filled with reactants, gloves are worn for placing the
plates into the electrophoresis chamber and
adjusting the wicks; they are also worn
for removing the plate after the run is
completed. After removal from the chamber, the plate is placed in a metal holder!
(Fig. 1) which permits reading the reactions without touching the plate or gel;
it also protects the reading box from the
bottom of the plate. The entire holder containing the plate may be kept in a large
petri dish if the test is to be held for additional readings. The holder is decontaminated by autoclaving. T h e wicks are decontaminated by autoclaving, and the buffer is aspirated from the chamber into a
J Designed by Miss Pinkie Gee, Research Assistant III, and Mr. Andrew Pasqual, Carpenter I,
California State Department of Public Health,
Berkeley, California.
April 1972
SAFETY PRECAUTIONS FOR HAA TESTS
529
FIG. 1. P l a t e holder
used in reading immunoelectroosmophoresis tests
tor hepatitis-associated
antigen or antibody.
closed flask and then autoclaved. The
chamber is then decontaminated by sterilization in ethylene oxide.
Staining the reactions entails the same
risks of contamination mentioned above
for immunodiffusion tests, and in the experience of this laboratory, staining has not
measurably increased the sensitivity of the
IEOP test for detecting HAA in test sera.
In addition to observing microbiologic
safety precautions, laboratory workers performing IEOP tests should be aware of the
electrical hazards involved in the use
of electrophoresis equipment, 10 and they
should be completely familiar with the
equipment before using it. Safety measures
for grounding and enclosing electrophoresis equipment have been described.10
Complement Fixation Tests, Microtiter
Method. Disposable plastic plates should
be employed, since it is uncertain whether
permanent lucite plates can be properly decontaminated to inactivate hepatitis viruses.
After the microdiluters have been used
to prepare serial dilutions of test serum,
they are blotted on paper contained in a
small, metal discard pan before they are
used for the next series of dilutions; more
blotters can be added to the pan as needed,
and after the run is completed these are
left in the pan and autoclaved. When the
serum dilutions are completed, the microdiluters are carefully placed in a pan containing water and decontaminated by boiling for 30 min. The beakers containing the
saline diluent used for pre-wetting the diluters and the distilled water used for rinsing them are placed directly in a discard
pan, without emptying the contents, and
are autoclaved.
The plates are not sealed with tape for
the incubation period after the addition of
complement; this is to avoid the production of aerosols and the splashing of infectious materials which may occur during
removal of a tape seal for addition of sensitized cells to the test. Instead, a nonabsorbent cardboard cover the size of the
plate is placed over the cups, and each
plate is wrapped in a plastic film's; this
serves to contain infectious materials if the
plate is accidentally tipped and the contents spilled. The plastic film also protects
the refrigerator, mechanical shaker, and incubator from infectious materials which
may contaminate the bottom of the plate.
§ Saran wrap, Dow Chemical Company, or Vitafilm, Goodyear Company.
530
SCHMIDT AND LENNETTE
After the incubation period for fixation of
complement, the wrapping is laid back from
the top of the plate, the cardboard cover
is removed and placed in a discard pan for
autoclaving, and the sensitized erythrocyte
suspension is added to the test. The plates
are then sealed with a tape cover to contain the contents of the cups during shaking, and the plastic film is drawn over the
top of the plates. The wrapped plates are
agitated on a mechanical shaker to disperse
the sensitized cells and the tests are incubated in an incubator at 37 C. until the
controls show the proper degree of hemolysis.
Discussion
Human hepatitis viruses have not yet
been propagated in a laboratory host system, and therefore it is impossible to monitor for infectivity of materials by conventional methods and to determine the efficacy of decontamination procedures. Available information on the sensitivity of hepatitis viruses to physical and chemical agents
is based upon early experiments using
human volunteers, and these studies indicated that hepatitis viruses, and particularly the "serum" hepatitis agent, are usually resistant to inactivation. 8 There is no
evidence that commonly used antiseptics
such as alcohol, ether, and zephiran inactivate the viruses, but heat sterilization,
either boiling for 30 min. or autoclaving
at 121 C. (15 lb. of pressure) for 20 min.,
has been effective in practice. Sterilization
of instruments and equipment in ethylene
oxide also appears to be effective in practice, as there are no reported cases of hepatitis attributable to instruments or hospital
equipment sterilized in this manner.
All blood specimens, even those negative
for HAA, should be considered potentially
infectious during handling in the laboratory. The "infectious" or short-incubation
A.J.CP.—Vol.
57
form of hepatitis is caused by an agent distinct from the one producing "serum" hepatitis and is not associated with HAA; this
agent is present in blood collected during
the acute phase of infection and may be
transmitted by either the oral or the parenteral route. 8 Further, dilution studies have
shown that blood specimens containing low
levels of HAA, undetectable by current
methods of assay, are infectious and may
transmit hepatitis to recipients. 8
In the past few years increasing numbers
of instances in which hepatitis with "Australia" antigenemia has occured in hospital
or laboratory personnel in the absence of
known parenteral injection with blood
or blood products have come to light.
This emphasizes that infection may occur
through nonparenteral exposure, and illustrates the need to employ strict microbiologic safety precautions for handling
blood and blood products.
References
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2. Gocke DJ, Kavey NB: Hepatitis antigen. Correlation with disease and infectivity of blooddonors. Lancet 1:1055-1059, 1969
3. Krugman S, Giles JP: Viral hepatitis: New
light on an old disease. JAMA 212:1019-1029,
1970
4. Krugman S, Giles JP, Hammond J: Infectious
hepatitis: Evidence for two distinctive clinical,
epidemiological and immunological types of
infection. JAMA 200:365-373, 1967
5. London WT, DiFiglia M, Sutnick AI, et al:
An epidemic of hepatitis in a chronic-hemodialysis unit. Australia antigen and differences in host response. N Engl J Med 281:571578, 1969
6. Murray R: Viral hepatitis. Bull NY Acad Med
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hepatitis-associated "Australia" antigen and
antibodies. J Immunol 105:604-613, 1970
9. Shulman NR: Hepatitis-associated antigen. Am
J Med 49:669-692, 1970
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