4. LABORATORY PROCEDURES

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4.1 GENERAL

The work location or work environment should be suitable for laboratory work, and have appropriate security to avoid unauthorised access to the laboratory. The work environment should also ensure minimal work-related injury to employees and visitors.

Lack of space or inappropriate equipment must not be a limiting factor for quality in culture or analysis.

4.1.1 REFERALS

See Appendix 1 for indications for referral to a cytogenetic laboratory. The laboratory should have policies for onward referral where cases require specialised expertise not provided locally e.g. chromosome breakage analysis, molecular genetic testing.

4.1.2 STANDARD OPERATIONAL PROCEDURES (SOPs)

Standard operational procedures, for techniques or use of equipment, should exist for all operational procedures in the laboratory. They should be written in a language understandable for the staff. SOP's should be updated annually. Obsolete versions of SOPs should be kept for at least 5 years. It is the responsibility of the laboratory director to ensure that all staff are appropriately trained, and have knowledge about and understand the standard operating procedures.

4.2 EQUIPMENT AND FACILITIES

Essential equipment should be serviced annually. All equipment and facilities in the laboratory should fulfil the requirements for the European Community (CE approved). Council Directive 93/68/EEC: 1993

To minimise equipment failure, all essential equipment should be duplicated (i.e. two incubators, two centrifuges, etc.). If any essential equipment is not duplicated for any reason, the laboratory should have a written "crash plan" on how to overcome failure affecting the laboratory work.

4.2.1 SAFETY CABINETS

All fresh cytogenetic samples are at risk of carrying dangerous pathogens e.g. Hepatitis B positive blood samples. Appropriate safety cabinets should be used for the containment of biological material, see the EC Directive (93/88/EEC). Horizontal laminar flow cabinets should be avoided as they offer no protection for the worker. Many countries have National regulations for the protection of workers, samples and the environment. If no National regulations exist it is recommended to consult documents as:- EC Directive (93/88/EEC), HSC, Advisory Committee on Dangerous Pathogens, The management and design and operation of microbial containment laboratories (ISBN 0717620344) or ZKBS advisory committee in Germany.

4.2.2 INCUBATORS

All incubators and other critical equipment should be fitted with an alarm or an override system to protect against malfunction of temperature and CO2 (where used) controls. It is recommended that centrally monitored alarm systems are available.

Laboratories performing prenatal analyses should possess at least two incubators for splitting of prenatal specimens. It is also recommended that prenatal and non-prenatal cultures are incubated separately to minimise the risk of microbial cross-contamination.

4.2.3 IMAGE CAPTURE SYSTEMS

To maintain a high quality service provision all image analysis systems should be maintained regularly with software upgrades.

The number of image processing systems should not be a limiting factor in specimen analysis. When using image analysis systems, one part of the analysis process, either the initial analysis or the checking, should be completed using a microscope to ensure small markers or additional chromosomes have not been overlooked.

To avoid unnecessary delays due to image systems faults/failure, a service agreement is highly recommended.

4.3 TECHNICAL ASPECTS OF CYTOGENETICS

4.3.1 CELL CULTURES

Duplicated or independently established cultures, where possible, are recommended for all types of cultured specimens.

4.3.2 BANDING

All karyotyping should be carried out using a banding technique. In no cases, except some chromosome breakage syndromes, should a report be issued without cells having been subjected to full analysis of the banding pattern for the whole chromosome complement.

The ISCN defines five levels of banding. This can be used as a guide for establishing the degree of resolution required in producing a result. Several useful methods have been developed to help assess banding quality. Some countries e.g. Germany and the UK use an alternative approach that designates a quality score representing which chromosome bands are visible at 250 (QAS 2), 350 (QAS 4) and 550 (QAS 6) bphs resolution. More information can be found on the ACC (www.cytogenetics.org.uk under info) and BVDH (www.gfhev.de/en/membership/ under quality management) websites.

Numerical and structural abnormalities have to be excluded at a banding level appropriate to the referral reason. One or more objective and reproducible method(s) must be used to assess banding resolution and must be described in the laboratory protocol book or User Guide.

A standardised method for assessing banding quality should be used, with an agreed minimum standard that may vary depending on the reason for referral. Full analysis must be completed to the satisfaction of the supervisor that numerical and structural abnormalities have been excluded to a level appropriate for the referral reason. Specific standards for resolution should be appropriate to the case and the type of tissue studied. The 400 bphs (QAS 4) level is the minimum level of resolution for studies to establish common aneuploidies in constitutional cytogenetics. A 550 bphs (QAS 6) level should be the minimum standard for referrals of mental retardation, birth defects, dysmorphic children or couples with recurrent pregnancy loss.

Where it is not possible to achieve the minimum quality for the referral reason, and no clinically significant abnormality is detected, the report should be suitably qualified whilst not encouraging repeat invasive procedures when these are NOT clinically justified.

4.4 ANALYSIS

4.4.1 KARYOTYPING/CHROMOSOME ANALYSIS

In general, a minimum of 5 cells should be fully analysed for constitutional analysis and 10 cells for a haematological analysis. If metaphase analysis involves a comparison of every set of homologues (including X & Y), band by band, then a minimum of 3 metaphases can be fully analysed. If one of the homologue pair is involved in an overlap with another chromosome the pair of homologues should be independently scored to ensure there is no structural rearrangement.

Additional cells may be counted depending on laboratory policy. An extended analysis and/or cell count is warranted when mosaicism is clinically indicated or suspected. The laboratory should have written protocol for the analysis criteria.

Hyper- and hypodiploid and polyploid cells should be fully analysed in constitutional and haematological analysis.

All cases should have an image or a slide stored for later review (see section 11.12.1).

Refer to the current ISCN for the definition of a clonal abnormality.

4.4.2 CHECKING

Checking of all cases by a second qualified cytogeneticist is essential. A senior supervisor or an experienced cytogeneticist should check the analysis.

4.5 INTRODUCTION OF NEW LABORATORY PROCEDURES

A laboratory should, when starting any new diagnostic service, have a protocol for training staff and testing new equipment so patients are not at risk from inappropriate handling of equipment/slides etc. One way of doing this is to divide the samples, and send half to an experienced laboratory until the necessary level of competence is achieved. Validation and SOPs of these procedures is required.

4.5.1 USE OF MOLECULAR TECHNIQUES

When molecular genetic techniques are more sensitive than conventional cytogenetics, they should be used once the method has been validated, (e.g. for Angelman or Prader Willi syndrome, other microdeletion syndromes, Fragile X syndrome, etc). This could result in onward referral of cases if a laboratory is unable to undertake such analysis. Exclusion of other chromosome abnormalities may still be required in most cases.

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Last changed: 2008-02-05