Advisory Committee on Dangerous Pathogens

Revised Advice on Laboratory

Containment Measures for work
with Tissue Samples in Clinical
Cytogenetics Laboratories
Supplement to: ACDP guidance on
protection against blood-borne infections
in the workplace: HIV and hepatitis

REVISED ADVICE ON LABORATORY CONTAINMENT MEASURES

FOR WORK WITH TISSUE SAMPLES IN CLINICAL CYTOGENETICS
LABORATORIES
Introduction

1. As a result of the re-assessment of risk associated with the culture

of known or suspected HIV infected samples, revised measures for
containment are now recommended. This revised guidance is issued
following a risk assessment undertaken by a Working Group
comprising representatives of the Association of Clinical
Cytogeneticists and the Royal College of Pathologists. Their
recommendations for revised containment measures in clinical
cytogenetics laboratories have been accepted by the Advisory
Committee on Dangerous Pathogens (ACDP), and are set out in the
following paragraphs.
2. This revised guidance represents what is considered to be good
practice by members of the ACDP. Following this guidance is not
compulsory, and it is open to individuals to take other action, but if
this guidance is followed it will normally be sufficient to comply with
the law. Health and safety inspectors seek to secure compliance with
the law and may refer to this guidance as illustrating good practice.

Work in
cytogenetics
laboratories

3. The advice in the following guidance applies to work in clinical

cytogenetics laboratories.
4. Cytogenetics involves microscopic analysis of chromosome
preparations derived from cells in the metaphase stage of mitotic division.
In vitro cultivation of cells forms an integral part of the methodology.
The processing of samples for analysis can be summarised as:

The sample is received and, where necessary, appropriate cell types

selected;

The tissue is set up in culture. Culture time will vary depending on

the sample type;

At the appropriate time, cultures are processed for cytogenetic

preparations (i.e. harvesting);

Cultures for harvesting are blocked in mitosis using a spindle

inhibitor, colcemid;

After a pre-determined time, cells are treated with a hypotonic

solution, fixed (3:1, methanol:acetic acid), and the mitoses spread on
clean glass slides;

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

After further processing with an enzyme, and staining to produce a

banding pattern, the chromosomes are analysed by light microscopy.

5. Most laboratories use a Class 2 microbiological safety cabinet for

sample and culture handling, in order to protect both the operator and the
sample from air-borne contamination. The glass front panel also affords
operator protection against contamination by droplets produced from
splashing. At various stages of processing, and especially during
harvesting, samples will need to be centrifuged.
6. Although all dividing cells can be processed for cytogenetic analysis,
the cells and tissues that are routinely cultured for diagnostic purposes are
discussed in the following paragraphs.
Current ACDP
guidance

7. General guidance on safe working procedures applicable to all persons

at risk of exposure to blood-borne viruses (BBVs) as a result of their
occupation are set out in Part 3, paragraphs 83-88 of the ACDP
publication Protection against blood-borne infections in the workplace:
HIV and hepatitis, 1995, HMSO ISBN 0 11 321953 9, (ACDP, BBV
guidance), to which this revised guidance forms a supplement. The
general guidance remains extant.
8. Guidance on laboratory work with blood-borne viruses is contained
in Part 4, paragraphs 116 181 of the ACDP, BBV guidance. In
particular paragraph 142 advises that where materials that contain or may
contain BBVs are being examined for purposes other than propagation or
concentration of virus, Containment Level (CL) 2 is acceptable if
additional precautions are taken. (i.e. CL2+). Paragraph 152 states that,
for the purposes of propagation or concentration in the laboratory, work
must be conducted at CL3.
9. Paragraph 156 goes on to advise that the cultivation of all cells from
known or suspected cases of HIV or other primate (human or simian)
retrovirus infection, must be conducted at Containment Level 3.
However, subject to the assessment of risk required under the COSHH
Regulations, permissive or mixed cells from those not known or suspected
to be infected with retroviruses may be handled at CL2+. The guidance
also points out that with prolonged incubation (e.g. beyond 3-4 days)
there is increasing likelihood of significant HIV production, and a local
risk assessment is necessary in each case, which should take account of the
potential risks from HIV and other BBVs that may be present.
These recommendations have been reviewed following a re-assessment
of the risks, as mentioned in paragraph 1 above, and annexed as an
appendix to this supplement. The revised guidance is set out in
paragraphs 11.2-11.7 below.

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

10. Paragraphs 131 - 132 give advice on sample labelling requirements.

There remains a need for known or suspected high risk of infection
specimens to be appropriately labelled in order that laboratories can make
a local risk assessment and apply local precautions, to ensure the health,
safety and welfare of staff.
For purposes of emphasis the relevant requirements for labelling of
high risk samples are set out in paragraphs 13-15 below, together with
a reminder of the legal obligation to label risk of infection samples
appropriately, under the Health and Safety at Work Act and COSHH
Regulations.
Revised
recommendations
for containment
in clinical
cytogenetics tissue
culture

11.1 In general, in clinical cytogenetics work, procedures should be

undertaken at Containment Level 2+* because of the ever-present risk of
contamination of samples with BBVs.
11.2 HIV risk samples
(i) The cultivation of lymphocytes (using PHA stimulation), and
other permissive cells, with CD4 receptors, from known or
suspected cases of HIV infection may be conducted at
Containment Level 2+, provided the incubation of cultures
does not exceed 100 hours.
(ii) The cultivation of bone marrow from known or suspected
cases of HIV infection may be conducted at Containment
Level 2+, provided incubation of cultures does not exceed 100
hours.
(iii)

Where incubation of lymphocytes or other lymphoid

tissue and other permissive cells from known or suspected
cases of HIV infection exceeds 100 hours, work should be
conducted at Containment Level 3.

(iv) The cultivation of chorionic villus cells, amniotic fluid cells,

fibroblasts or other solid tissue cell types and non-lymphoid
tumours in clinical cytogenetics, from known or suspected
cases of HIV infection, may be conducted at Containment
Level 2+, irrespective of the period of culture. This applies
equally where the initial sample is contaminated with blood.
(v) For any procedure proposed using material from known or
suspected cases of HIV infection, which does not fall within
the above categories, a separate risk assessment will need to be
undertaken.
*

NB: Throughout the document, Containment Level 2+ requires that a Class 1

or Class 2 microbiological safety cabinet should be used.
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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

11.3 Hepatitis B risk samples

The cultivation of all cells (except liver cells) from known or suspected
cases of HBV infection may be conducted at Containment Level 2+. If,
very exceptionally, a liver cell culture is to be performed, a separate risk
assessment should be undertaken.
11.4 Hepatitis C risk samples
The cultivation and processing of all cells from known or suspected cases
of HCV infection may be conducted at Containment Level 2+.
11.5 All other routine samples not suspected to present a risk
All other samples may be handled and cultured at Containment Level 2+,
provided they are not suspected of being infected with any other Hazard
Group 3 or 4 pathogen.
11.6 Samples at risk of being infected with Hazard Group 3 or 4
pathogens other than HIV, HBV and HCV
A local risk assessment is necessary in all unusual cases. If significant risk
of infection is shown, the appropriate Containment Level should be used.
11.7 Lymphoblastoid cell lines and other continuous lines of
susceptible cells
In general, continuous lines of susceptible cells, including lymphoblastoid
cell lines, may be handled at Containment Level 2+ unless, on the basis of
a risk assessment, it is considered that they should be handled at a higher
Containment Level.
Specifications for
Containment
Level 2+ in
cytogenetics

12.1 In addition to the measures and facilities normally specified for

work at Containment Level 2, the additional measures listed below should
also apply when Containment Level 2+ is specified. These extra
precautions should be in constant use to guard against percutaneous
inoculation, contamination of the skin, mucous membranes and working
surfaces.

Work must be conducted in a designated area of the laboratory with

sufficient space to work safely. Access of unauthorised persons to the
work must be prevented, to ensure that the person conducting the
work is free from the risk of disturbance or accidental contact with
others. An appropriately sited Class 1 or 2 microbiological safety
cabinet would meet this requirement, and is recommended for
culturing and processing samples in clinical cytogenetics laboratories
and see paragraph 12.2 below.

The use of sharps and glassware must be avoided wherever possible.

Where such use is essential, particular care must be taken in their
handling and disposal after contact with unfixed materials.

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

Lesions on exposed skin should be covered with waterproof dressings.

Protective clothing must include a laboratory coat, disposable gloves

and other personal protective equipment appropriate to the task.

The workstation must be cleared of unnecessary equipment.

Sealed rotors or buckets should be used for the centrifugation of

samples and unfixed cell suspensions. In the event of breakage, the
rotor or bucket should be opened in a Class 1 or 2 cabinet.

The bench surface and any equipment must be disinfected

immediately after completion of work.

A suitable disinfection policy must be in operation.

Full consultation with staff in respect of containment must take place,

and protocols for the safe conduct of the work must be agreed and
strictly adhered to.

The need for immunisation of laboratory staff with HBV vaccine,

together with follow-up procedures, in line with the advice contained
in paragraphs 87-88 of the BBV guidance, should be considered.

12.2 The use of microbiological safety cabinets

Containment Level 2 specifies that although work in general may be
conducted on an open bench, for vigorous shaking or mixing a Class 1
microbiological safety cabinet, or other equipment designed to contain
resultant aerosols and droplets, must be used. However, as BBVs are
unlikely to be infectious by the airborne route, the use of a Class 2 cabinet
for this purpose is acceptable when protection of the work from
contamination is essential.
Labelling of
samples

13. It is important that known or suspected high risk of infection

specimens are appropriately labelled. This will allow laboratories to make
a suitable local risk assessment, and apply relevant local precautions.
14. The ACDP made a clear recommendation in the BBV guidance, to
which this forms a supplement, (see paragraphs 131-132) about the
labelling of high risk specimens with regard to the culturing of BBV high
risk samples. Furthermore, in order to comply with existing HSAC
guidelines and legislative requirements, the use of danger of infection
labels for specimens known or suspected to carry a BBV is strongly
recommended.

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

15. For emphasis, the earlier recommendations are (in part) reproduced
below.
Anyone responsible for taking and/or despatching
specimens or other potentially hazardous material for
laboratory examination has duties under the Health and
Safety at Work etc. Act (HSWA) and the COSHH
Regulations to conduct the work safely. One specific
requirement (covered by Section 3 of the HSWA) is to
convey knowledge of known or suspected hazards to those
who need to handle any material that is sent for
examination, and this may be achieved by inclusion of
clinical details on the request form which accompanies
the specimen. Such information however is confidential
and should not be readable by persons handling the
specimen while it is still contained in its packaging.
Use of some form of danger of infection label; (e.g. a
yellow biohazard sticker) is appropriate for labelling
specimens known or suspected to come from individuals
with BBV infections. The specimen container should be
labelled on the outside and be clearly visible. The
accompanying paperwork should also be appropriately
labelled. It is good practice for those requesting tests to
provide as much information as is relevant, consistent
with maintaining patient confidentiality, with any request
for a laboratory investigation.

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

APPENDIX
ASSESSMENT OF RISK OF PROPAGATION OF HIV IN TISSUE CULTURE IN
CYTOGENETIC LABORATORIES
RISK ASSESSMENTS FOR INDIVIDUAL SAMPLE-TYPE CULTURES

1. Lymphocyte cultures: Approximately 0.51ml. heparinised whole

blood is added to 10ml. culture medium containing phytohaemagglutinin
(PHA), a mitogen which stimulates T-cell lymphocytes to divide.
Cultures are harvested at either 48 hours or 72 hours after initiation.
Whilst the majority of blood samples processed in this way come from
adults and children, fetal cord blood obtained in utero or at delivery can
also be cultured in this way.
Risk assessment
There have been concerns that this
lymphocyte culture system might propagate HIV to dangerously
high levels. However, experiences with routine culture in virology
departments has indicated that this is not the case. Using culture
conditions optimised for production of HIV (PHA stimulated
blood supplemented with interleukin 2, with the addition of a
feeder lymphocyte layer), it has been found that little or no virus
production, measured by retrovirus specific reverse transcriptase
activity and levels of p24 core antigen, occurs within 100 hours,
and that significant viral production takes 10-17 days. The
conclusion, therefore, is that there would be no HIV production in
routine lymphocyte cultures in clinical cytogenetics, provided these
do not exceed 100 hours.
Plasma from patients with a high viral load at the time of
seroconversion when terminally ill with HIV infection would have
a titre of between 103 and 107/ml. plasma. These samples are
currently handled in biochemistry and haematology laboratories at
CL2+. In clinical cytogenetics there would be approximately 1:10
dilution of the viral concentration when the sample is set up in
culture. As there is no significant increase in the viral load during
culture, the subsequent processing would not present a greater risk
than activities in other laboratories handling HIV samples.
Furthermore most harvesting of samples in UK cytogenetics
laboratories occurs under exhaust protection and splash and droplet
protection in Class 1 or 2 cabinets, by staff highly trained in aseptic
techniques.

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

2. B-cell lymphoblastoid cell cultures: Immortalised cell lines, derived

from B-cell lineages are established by transforming cells with Epstein Barr
Virus. These can grow indefinitely, and are often held in long-term
storage facilities using liquid nitrogen. Changes in technology in recent
years have meant that these are initiated less frequently than in previous
years, and they are used now more in research than in diagnostics.
Risk assessment
There is evidence that some B-cells
transformed with Epstein Barr Virus may support HIV-1
replication when experimentally exposed to T-cell-line adapted
laboratory strains of HIV-1 and HIV-2. However, there is no
evidence of lymphoblastoid cell lines derived from known HIVpositive donors being HIV positive. Although B-cells are less
efficient at HIV propagation than T-cells, because of the length of
time in culture the viral titre could eventually be high. Continuous
lines of susceptible cells and lymphoblastoid cell lines can be
handled at CL2+ unless a risk assessment shows significant risk of
production of HIV. Such cell lines should not be distributed
within the scientific community unless they have been tested and
shown to be uninfected, or the receiving laboratory acknowledge
their status, in writing, before receipt.
3. Amniocyte cultures: Amniocentesis is the standard procedure for
cytogenetic analysis of the fetal karyotype. Usually performed midtrimester, 15-20ml. amniotic fluid is removed under ultrasound guidance,
and the various types of fetal and amnion cells contained therein are
removed by centrifugation and set up in culture. Culturing usually takes
7-14 days, although this period may be longer if large numbers of cells are
required for molecular or biochemical diagnosis.
Risk assessment
There is no evidence that amniotic fluid
cultures support the sustained production of HIV, and consequently
there appears to be no risk of concentrating the virus by routine
culture techniques. Blood stained amniotic fluid samples from HIV
infected patients do not present any additional risk of propagation of
HIV, because the lymphocytes within the blood require specific
mitogens for transformation. The majority of blood cells are usually
removed after a few days, when the tissue culture medium is
replaced with fresh medium. However, as some residual infectivity
may remain, care should be taken throughout to avoid percutaneous
injury. Great care should be exercised in handling glass coverslips
when in situ culture techniques are employed.
4. Chorionic villus samples (CVS): Biopsies from the placenta are
usually taken in the first trimester, from 10 weeks gestation onwards.
Direct preparations of the trophoblast will yield spontaneously dividing
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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

cells which, although of poor quality, can be used to diagnose many of the
common cytogenetic abnormalities. Direct preparations do not require
culturing, although sometimes the tissue may be placed in an incubator
overnight prior to processing. CVS however are also usually cultured to
improve diagnostic accuracy. Cells derived from the mesenchyme core of
the villus are cultured in a way similar to amniocytes. A CVS sample
needs to be manually processed on receipt, in order to remove any
maternal decidua or clotted blood adhering to the villus. This is achieved
most effectively by the use of sharp needles.
Risk assessment
There is no evidence that CVS cultures
support the production of HIV. While any associated macrophages
may be infected with HIV, these cells do not grow well under
routine culture conditions.
However, the initial sample, particularly if contaminated with
blood from a patient infected with HIV, could present a risk during
processing. There is epidemiological evidence to show that the risk
from percutaneous injury with a hollow bore needle is greater than
with a solid needle. For example, needlestick injuries in health care
workers, involving hypodermic syringes, are associated with a
greater risk of HIV transmission than injuries in surgeons using
suture needles. In addition, there is experimental evidence to show
that there is a significantly greater wiping effect (90%) through
wearing disposable latex gloves in percutaneous injuries involving
solid needles compared with hollow bore needles. This reinforces
the need to wear disposable protective gloves when handling and
processing samples, and to avoid the use of sharps wherever
possible. When the use of sharps is essential, particular care must
be taken, and solid needles should be used for dissection rather
than syringe needles.
In HIV high risk cases, the use of plastic pipettes should also be
considered for manipulating the specimen, where the condition of
the sample allows it. Great care should also be exercised in
handling glass coverslips when in situ culture techniques are
employed.
5. Solid tissue samples: Tissue biopsies and post mortem samples are
grown for cytogenetic, biochemical and molecular analysis from a variety
of sources, and also stored as cell lines. Common samples used in
diagnostics include tissues from early products of conception, fetal tissues
(e.g. skin, fascia lata, pericardium, muscle, amnion, chorion etc.), and skin
biopsies from neonates, children and adults. Cells are often in culture for
3-4 weeks.

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

Risk assessment
There is no evidence that fibroblast
cultures support the sustained production of HIV. Whilst
Langerhans cells in skin samples may contain HIV, the cells do not
grow well under routine culture conditions. The associated
keratinocytes are unlikely to be susceptible. However, the initial
sample, particularly if contaminated with blood from an HIVinfected patient, could present a risk during processing. As with
CVS samples, when the use of sharps is essential, particular care
must be exercised in their handling and disposal.
6. Marrow samples: Diagnosis and prognosis of leukaemia patients is
assisted by cytogenetic analysis of marrow cells. Although some analysis is
done on direct preparations, where culturing time is limited by the action
time of the spindle inhibitor, colcemid, most laboratories use a range of
culture times from overnight to 48 hours incubation, and occasionally as
long as 72 hours. In some lymphoid disorders, a B-cell mitogen may be
used. Leukaemia patients may be immunosuppressed, increasing chances
of infection. Adult T-cell leukaemia may, rarely, be caused by HTLV1, but
the prevalence of HTLV1 infection in the UK is thought to be very low.
Risk assessment
It is very unlikely that there would be any
significant production of HIV in the culture regimens normally
employed with marrow samples.
7. Solid tumour samples: Samples from patients with other forms of
cancer are increasingly being analysed using cytogenetic techniques.
Although some malignant tissue, such as lymphomas, contain
spontaneously dividing cells, long-term cell culture of 10-60 days is also
necessary in most cases.
Risk assessment
It is most unlikely that cultures established
from solid tumours would be a productive source of HIV. Shortterm cultures established from lymphomas present no significant
risk. However, a separate risk assessment should be made in each
case that may potentially involve permissive cells. Immortalised cell
lines derived from lymphoid cells should probably be subject to the
same containment conditions as lymphoblastoid cell lines, and
handled at CL2+. In samples from patients presenting with
lymphadenopathy, the possibility of HIV, or HTLV1 infection
being the cause should be considered.

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Supplement to ACDP guidance on protection against blood-borne infections in the workplace: HIV and hepatitis

If you require further FREE copies of this publication

please contact:
Department of Health
Skipton House (Room 6308)
Mr T Hearn (ACDP Secretariat)
80 London Road
London
SE1 6LH
Fax: 0207 972 5155
Tel: 0207 972 5339
E-mail: [email protected]
It is also available on our website at:
www.doh.gov.uk/ACDP

First Published: July 2001

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