5. SPECIFIC CHROMOSOMAL ANALYSIS

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5.1 PRENATAL

5.1.1 AMINOCYTE CULTURES

To minimise the risk of contamination, or culture loss due to incubator failure, duplicate cultures should preferably be handled separately, kept in separate incubators, if possible, running on a different electrical circuits. Prenatal cultures should be maintained with two different cell culture media, or with different batches of the same cell culture media and other reagents. The possibility of maternal cell contamination, pseudomosaicism, true mosaicism and in vitro aberrations must be recognised and the system of culture and analysis used designed to detect and differentiate these problems.

Harvesting or subculturing of all cell cultures from an individual sample together should be avoided.

If possible back up cultures should be kept until the final report is written.

Facilities should be available for freezing viable cells, e.g. for unresolved cases of abnormal foetal pathology.

5.1.2 CHORIONIC VILLI CULTURES

Before a CVS sample is cultured it must be dissected and maternal decidua separated from the villus to reduce the chance of maternal cell contamination. It should be clear from the referral form whether the sample has been dissected or not prior to its' arrival in the laboratory. If an initial cytogenetic diagnosis is made on short-term preparations, a long term culture should be available for confirmation, in order to minimise problems of interpretation (Eucromic 1997, ACC Collaborative study, 1994). Analysis solely on short-term incubation preparations (direct preparations) is not recommended (Eucromic 1997, ACC Collaborative study, 1994). If the sample is of an inadequate size for both short and long term cultures, analysis from a long term culture is recommended.

5.1.3 FOETAL BLOOD CULTURES

The foetal blood sample should be checked to ensure it is not mixed with maternal blood, and originates only from the foetus. Several haematological methods are available. (Alkaline Phosphatase, Kleinhauer, Coulter counter sizing). Both foetal blood and amniotic fluid samples should be analysed unless there is a valid reason not to do so e.g. abnormal foetal blood result and pregnancy terminated.

5.1.4 MOSAICISM IN PRENATAL STUDIES

Two or three cultures should be set up for each sample. Analysis of a second or third culture is essential in cases of suspected mosaicism or pseudomosaicism e.g. trisomy 2 or where the abnormality is not consistent with continued fetal development (see Hsu et al., 1996, 1997). In general, if the same abnormality is present in two independent cultures, mosaicism is confirmed.

For in situ preparations, analysing cells from one cell culture may be sufficient if not all from the same colony. However, it is recommended that at least two independent cultures are established to be able to rule out pseudomosaicism. When sufficient colonies are available, no more than two cells should be counted and analysed from a single colony (except when excluding a single cell anomaly). If colonies are insufficient for this to be achieved, a comment should be made in the report. It is unreasonable to expect all cases of true foetal chromosome mosaicism or small structural rearrangements to be detected by a routine level of analysis.

A written procedure for delineating different types of mosaicism should be drawn up for guidance within the laboratory. Individual cases can require careful assessment and discussion and the number of cells counted and analysed may exceed the minimum.

In an amniotic fluid culture, detection of a mosaicism must be followed up by extensive examination of cells from an independent culture, or from independent colonies. Failure to confirm the abnormal cell line provides reassurance of a normal pregnancy but, depending on chromosomes involved and the nature of the abnormality, supplementary investigations may be appropriate (see Hsu et al., 1996, 1997). To facilitate the elucidation of mosaicism and in vitro abnormalities, the independent colony method is recommended.

In CVS, the significance of mosaicism may depend on the distribution of the abnormality amongst different cell types in direct and cultured preparations, and the chromosome/s involved (Eucromic 1997, ACC Collaborative study, 1994).

The possibility of foetal uniparental disomy in some cases cannot be ignored, and additional tests may be required to resolve uncertainty. UPD studies should be considered where there is mosaicism or confirmed placental mosaicism involving chromosomes 7, 11, 14 & 15 and in homologous and non-homologous Robertsonian translocations involving 14 & 15, and markers chromosomes of chromosome origin 7, 11, 14 & 15 (Kotzot 2002; Robinson et al., 1996; ACC Prenatal Guidelines, 2005).

5.2 POSTNATAL

5.2.1 POSTNATAL CHROMOSOME ANALYSIS OF BLOOD AND SOLID TISSUE/PRODUCTS OF CONCEPTION.

See ANALYSIS (Section 4.4.1). Extended analysis includes analysing a minimum of 30 cells is recommended when clinically relevant mosaicism is suspected (giving appropriate confidence limits using Hook's tables for lymphocyte cultures, Hook 1977). FISH analysis may be the most appropriate method of confirming suspected numerical mosaicism if a suitable probe is available. In some instances more than one tissue type should be investigated e.g. Pallister-Killian syndrome or trisomy 8 mosaicism.

Referring clinicians must be made aware that it is not possible to reliably exclude mosaicism from any analysis.

5.2.2 MOSAICISM

In cases where mosaicism may be expected to be present (e.g. sex chromosomes abnormalities or chromosome breakage syndromes), the number of

cells counted and scored should be sufficient to rule out mosaicism or clonality. An extended analysis is usually adequate (see 5.2.1). However, the laboratory should consider the common occurrence of age related sex chromosome losses and/or gains before reporting sex chromosome mosaicism (Guttenbach et al., 1995; Gardner and Sutherland, 2003). Laboratories should also be aware that the level of mosaicism may vary between tissues.

5.2.3 PRODUCTS OF CONCEPTION/FOLLOW UP SPECIMEN

Follow up of abnormal cases may form a part of internal quality control. However, if foetal morphology does not confirm the laboratory findings, foetal tissue samples should, where possible, be analysed.

5.2.4 CHROMOSOME INSTABILITY SYNDROMES

The rarity of chromosome instability syndromes and the interpretational problems associated with chromosome breakage syndromes requires that inexperienced laboratories refer such cases to laboratories with proven expertise. Classic breakage syndrome disorders include: Ataxia telangiectasia, Bloom syndrome, Fanconi anaemia, Nijmegen syndrome. Other syndromes involving defective DNA replication/repair (e.g. Cockayne syndrome and Xeroderma pigmentosum) are not amenable to cytogenetic methods of confirmation.

Clastogen studies should only be undertaken with appropriate negative control samples and, if available, positive control samples

All control and test samples should be collected, processed, cultured and harvested in parallel.

Controls should be appropriately matched (e.g. sex, age etc.). The patient and control samples should be analysed blind.

Sufficient numbers of metaphases must be examined in order to ensure that any chromosomal damage detected is significant.

• Bloom syndrome
  

  As some affected individuals have a population of cells with a normal SCE frequency, examination of 20 metaphases is advisable. The laboratory should have a record of the SCE frequencies found when the same methods are applied to a range of normal control samples.

• Fanconi anaemia
  

  Diagnosis and exclusion should be made by analysis in cultures exposed to clastogenic agents. Sufficient cells must be examined to exclude the possibility of somatic mutation, which is common in Fanconi anaemia. Analysis of at least 50 but preferably 100 metaphases is recommended. The efficacy of the clastogen used should be checked against either an untreated control or SCE levels in treated samples.

• Ataxia telangiectasia and Nijmegen syndrome
  

  The aberration frequency in irradiated cultures, scored from 50 to 100 metaphases, should be compared with normal control cultures. As some ataxia telangiectasia patients display an intermediate response to irradiation, screening of 50 banded metaphases for rearrangements, involving the T-cell antigen receptor loci on chromosomes 7 and 14, should also be carried out.

5.2.5 OTHER RARE SYNDROMES DETECTED BY CYTOGENETIC ANALYSIS

Despite recent advances in the understanding of the molecular basis of some disorders, cytogenetic studies are often the first step in making a diagnosis.

Sufficient numbers of metaphases must be examined in order to ensure that any chromosomal damage detected is significant.

• Roberts syndrome
  Fifty block (Leishman/Giemsa stained) or C-banded metaphases should be scored for paired centromeres, centromeres puffing and tramline chromosomes. Fifty banded metaphases should be counted, for evidence of aneuploidy.
• ICF syndrome
  Fifty banded metaphases should be scored for anomalies of the heterochromatic regions of chromosomes 1, 9 and 16 and for multi-branched configurations.

5.3 ONCOLOGY: LEUKAEMIAS AND SOLID TUMOURS

All laboratories offering a diagnostic service should be able to provide an analytical and interpretive service for a range of haematological disorders see Appendix 1. Referral can be at diagnosis, follow up after treatment, including transplantation, relapse/transformation or as part of a national or locally agreed trial.

5.3.1 BONE MARROW

In haematological and solid tumour cultures, the culture conditions should be optimised where possible by utilising direct, short term and synchronised cultures to improve the mitotic index. Laboratories should be aware that culture times may affect the detection of an abnormal clone. When B- or T-cell lymphoproliferative disorders are suspected, suitable mitogens should be added to additional cultures.

5.3.2 SOLID TUMOUR

Solid tumour cultures may require both multiple cultures and longer incubation (>72hours). It is recommended that the laboratory has previous experience in the tissue culture of various cell types before setting this up as a diagnostic service.

5.3.3 HAEMATOLOGICAL CHROMOSOME ANALYSIS

Sufficient number of cells should be examined to detect the presence of clonal evolution. The quality of metaphases obtained from unstimulated blood and from bone marrow samples is generally poor, particularly in leukaemia. As normal cells with better chromosome morphology may be present, it is important to analyse cells of varying quality in order to maximise the likelihood of detecting a clone. Abnormal cells are often those of poorer quality and sufficient cells should be analysed to establish the clonality of the abnormality (see ISCN for definition of clonality).

There is a high possibility of an abnormality being present in either a few cells or the presence of several subclones. When a normal karyotype is found, it is preferable that a minimum of 10 cells are fully analysed and a further 10 are screened for abnormal chromosomes for diagnostic samples, referral at relapse or transformation. If a sample yields fewer than twenty normal cells, the report should be suitably qualified.

For referrals where cytogenetic follow-up after treatment/remission is required the following analysis is recommended:

• If a normal result was obtained at diagnosis, further analysis is usually not appropriate.
• If abnormal result was obtained at diagnosis: a minimum of 20 metaphases should be scored for the relevant anomaly. In some instances, FISH may be appropriate for follow-up studies.
• For post-transplantation samples, a minimum of 30 metaphases should be scored for the presence or absence of the marker used to differentiate between donor and recipient cells e.g. the Y chromosome in mixed sex transplants. FISH may be more appropriate here also.

N.B. Definition of Scoring - To check for the presence or absence of a particular karyotypic feature in a number of cells

5.3.4 ONCOLOGY CHROMOSOME ANALYSIS

Adequate numbers of metaphases of varying quality should be analysed or examined before the report of a normal karyotype or of the existence of an abnormal clone is given. If a sample yields fewer than ten normal cells, the report should be suitably qualified.

Reporting and interpreting the results of tumour work is a specialised area, where close co-operation between the laboratory and the referring histopathologist is vital.

5.4 CGH AND MICROARRAY TECHNOLOGY

These methods are still considered to be experimental. However, any laboratory using these techniques in their clinical work should introduce SOPs. Clinical samples analysed according to these techniques should be treated as routine samples once an internal validation of the test has been established and handled according to appropriate laboratory guidelines.

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Last changed: 2008-02-05