GUIDELINES FOR CLINICAL CYTOGENETIC DIAGNOSTIC LABORATORIES IN BELGIUM

1. Introduction

Cytogenetic diagnostics has been performed in Belgium for over 30 years embedded within the Genetic Centres. While overall, the quality of cytogenetic diagnostics in Belgium can be considered excellent, several reasons prompted the need to create guidelines on the way of working in a cytogenetic laboratory and to create a framework in which to operate. These reasons are:

(1) Within the individual laboratories, often the guidelines created in neighbouring countries have been implemented. However, due to the specific customs of the Belgium people and due to the unique healthcare and reimbursement system in Belgium, often these rules have been bend and required specific adjustments within the individual laboratories. This has lead to (sometimes huge) differences in the way of working.

(2) It is anticipated that quality control systems will become more and more implemented possibly leading to a mandatory accreditation system (as is already in existence in neighbouring countries). Currently, the creation of rules is done within the individual laboratory which leads to redundant efforts and results in disharmonic rules in the different laboratories.

(3) Other medical disciplines with whom cytogenetics collaborate have often unreasonable or excessive expectations. The creation of rules will provide a framework with which cytogeneticists can communicate across medical disciplines.

(4) Patients may receive different diagnoses for a similar indication. This leads to confusion, both with medical practitioners as with lay people.

(5) Patients demand insights in test results and often question the laboratory practises. Harmonizing these practises will provide a legal framework and strengthen genetic diagnostic services in Belgium.

For these reasons, it was decided in 2004 within the Belgium Society of Human Genetics, to create a working group on cytogenetics to draft guidelines on the way of working in the diagnostic cytogenetic laboratory. Representatives of all genetic centres have participated in creating this document.

We believe this document will contribute to the quality and the harmonisation of cytogenetics in Belgium. Due to the rapid technical changes which are occurring, adjustments and improvements to this document will be required. We strongly believe that updating these guidelines will become a continuous effort for all who is concerned with cytogenetic quality.

2. Prenatal cytogenetic analysis

2.1. Prenatal chromosomal analysis

Prenatal chromosomal analysis can be performed on three types of tissues : Chorion vili, amniotic fluid and blood derived from the umbellical cord. Almost all chromosomal analyses are performed on (cultured) amniotic fluid and chorion vili cells and are discussed in this chapter.

2.1.1. Chorion vili

Before the start of the culture are the vili screened for the presence of maternal contamination. Potential maternal tissue is removed. Part of the villi are transported to the laboratory for FISH analysis (see xxx), while the other villi are put in culture.

Two types of analyses can be performed:

- Short term culture (24 hours) results in the analysis of cytotrophoblast cells.

- Long term culture results in the analysis of mesenchymal cells. The analysis occurs as described for amniotic fluid cells.

Since the long term culture has the highest risk for maternal contamination, it is advised to perform the short term culture.

The risk of confined plancental mosaicism in the CVS is estimated to be between 1and 2% while absent in amniotic fluids. Therefore, in general, for the detection of chromosomal anomalies we recommend prenatal diagnosis by amniotic fluid analysis.

2.1.2. Amniotic fluid

Amniotic fluid samples are preferentially brought in culture preferentially within 72 hours after sampling. Upon arrival of the sample in the laboratory, part of the cells are transported to the FISH laboratory for direct analysis of the amniotic fluid cells (see xx) and the supernatant is put aside to determine the alpha foeto protein concentration. The other fraction is brought into culture.

2.1.3. Culture

While either the in situ method, the flask method or a combination of both methods is in use, the in situ method is recommended as it is the fastest method and reduces the chance on false positives due to mosaicisms in the culture. At least, two independent cultures (with two different media) have to be initiated from each AF sample and microscopic analysis should be initiated from at least these two independent cultures. The double initiation is recommended in order to distinguish between mosaïcism and pseudomosaicism : analysis on both cultures should be done for each sample and has to be done in case of any anomaly in one of both cultures. It is suggested to start the two different cultures within two different incubators with independent electric and gas circuits. In case of a technical failure in one incubator, the other incubator can serve as a backup.

2.1.4. Analysis

For the in situ method, colonies can be distinguished and chromosomes are analysed from different colonies. Using the flask method, chromosomes analysed can be derived from the same original cell. Therefore, more analyses are required for the flask method compared to the in situ method. A distinction can be made between the counting (count the number of chromosomes), the analysis (determination that all chromosomes appear normal and are present in two copies, without making a karyogram) and making a karyogram. The banding resolution should be at least at the 400 band level. Below are the minimum requirements and between brackets the best practise requirements:

	Flask method	In situ method (different clones)
Total Counting Analysis Karyogram	15 (20) 15 (20) 10 (15) 2 (5)	10 (15) 10 (15) 5 (10) 2 (5)

Maximum time for reporting normal karyotypes is 21 days for amniotic fluid and CVS culture analysis.

2.1.5. Reporting

A written report must be produced for each sample, even no result could be obtained. A non ambiguous identification of patient and sample has to be made on the report. Items included in the report may vary considerably from a centre to another : for prenatal diagnosis, level of resolution and banding type and quality are not mandatory items in the report but can be included. Reporting pseudomosaicism and structural variants is not mandatory. However, technical and quality limitations (including limited number of metaphases, bad quality, …) should be added on the report.

2.2. Prenatal FISH analysis

2.2.1. CVS

- Karyotyping after short term culture is by most centers achieved 48 hours after sampling. An additional FISH procedure can be useful when not enough metaphases are obtained.
- If only long term CVS is performed, trisomy 21 screening can be performed to allow for rapid prenatal diagnosis and reduces anxiety for the pregnant women.
- Possibly XY screening can (but does not need to) be performed. This test enables some level of internal quality control. In case of male foetuses, the level of maternal contamination in the chorion villi can be determined.

2.2.2. Amniotic fluid

FISH on bloody amniotic fluid can lead to misinterpretation of the results and is discouraged.

With indications maternal age, psychological or increased triple test values:

- It is advised to perform trisomy 21 screening on all amniotic fluid samples in addition to the karyotype. This allows for rapid prenatal diagnosis and reduces anxiety for the pregnant women.
- Possibly XY screening can (but does not need to) be performed. This test enables some level of internal quality control and enables the evaluation of maternal contamination in case of a male foetus.
- Routine testing for chromosome 13 and 18 is discouraged. Good ultrasound practises will identify most cases where trisomy 13 and 18 screening is warranted.

With ultrasound indications

- When ultrasound anomalies are detected but no chromosome specific aneuploidy testing can be indicated, chromosome 21, XY, 13 and 18 screening is warranted.

- If based on the ultrasound indications a specific chromosomal aneuploïdy or microdeletion is suspected, a specific rapid test should be performed.

2.2.3. Criteria for interpretation and reporting

Currently, all centers use FISH on uncultured amniocytes to perform prenatal aneuploidy screening. Based on current practises, the following interpretation and reporting guidelines are drafted:

- 50 nuclei are scored by one technician

If in 45 or more nuclei two signals are detected, disomy for the tested chromosome is reported.

o If in 35 or more nuclei one or three signals are detected, monosomy or trisomy for the tested chromosome is reported.

o If intermediate values are detected a second analysis is performed and the scoring is performed by another analyst. A total of 200 nuclei are scored.

If in over 90% of the nuclei two signals are detected, disomy for the tested chromosome is reported.
If in over 70% of the nuclei, one or three signals are detected, monosomy or trisomy for the tested chromosome is reported.
In all other cases, the degree of mosaicism for the respective chromosomes is reported.
If only less than 200 nuclei are found, the FISH is performed on the cultured cells.

o If less than 25 nuclei are informative, no results are reported

o If between 25 and 50 nuclei are informative, the result is reported with a cautionary statement.

- The reporting is done within one week after sampling.