HOKLAS SC-35
Issue No. 3
Issue Date:20 July 2017
Implementation Date: 20 July 2017
Page 1 of 16

HOKLAS Supplementary Criteria No. 35

‘Medical Testing’ Test Category – Cytogenomics

1Introduction

1.1This document is an application document forthe requirements of HKAS 002 and HOKLAS 015 accrediting cytogenomic examinations within the test category of Medical Testing.This document sets out only those specific requirements which require further elaboration but does not include all the accreditation requirements. Therefore, it has to be read in conjunction with HKAS 002, HOKLAS 015 and HOKLAS SC-33.

1.2The checklists given in the Annex serve as guidance for laboratories to self-assess their management system and operation procedures against the requirements given in HOKLAS 015 and this document.

2Scope of accreditation

2.1HKASprovides accreditation under HOKLAS for cytogenomics in the following areas:

2.1.1Constitutional Cytogenetics (prenatal and postnatal)

2.1.2Cancer Cytogenetics (excluding solid tumours)

2.1.3Fluorescence in situ hybridisation (FISH)

2.1.4Chromosomal Microarray Analysis

2.1.4.1array comparative genomic hybridisation (array CGH)

2.1.4.2single nucleotide polymorphism array (SNP array)

3Personnel

3.1A qualified pathologist providing consultation and clinical interpretation for test results in any test areas ofcytogenomics shall have obtained the Fellowship of the Hong Kong College of Pathologists in an appropriate specialty plus qualification in relevant subjects in cytogenomicsfrom a university and/or professional body and two years' post fellowship full-time equivalent laboratory training in an ISO15189-accredited cytogenomic laboratory, or at least 5 years of laboratory experience in a laboratory providing the cytogenomic test services before 1 September 2008 (the launching date of HOKLAS for the accreditation of tests and examinations in Medical Genetics).

3.2A pathologist nominated by a laboratory to be its signatory for any test areas in cytogenomicsshall seek endorsement from the Hong Kong College of Pathologists that his/her training and experience meet the requirements of a signatory for such tests. It will be the responsibility of the pathologist to seek endorsement from the Hong Kong College of Pathologists and provide HKAS Executive with the written confirmation from the Hong Kong College of Pathologists.

3.3A medically qualified person nominated by a laboratory to be its signatory to provide consultation and clinical interpretation for test results in constitutional cytogeneticsand cytogenomics shall have obtained Fellowship in an appropriate specialty (e.g. Obstetrics & Gynaecology, Paediatrics) plus qualification in the relevant subjects in cytogenomics from a university and/or professional body and two years' post fellowship full-time equivalent laboratory training in an ISO 15189-accredited cytogenomic laboratory, or at least 5 years of laboratory experience in a laboratory providing the cytogenomic test servicesbefore 1 September 2008.

3.4A biomedical scientist reporting test resultsin constitutional cytogenetics without clinical interpretation shall be MLT Board Part I registered (or exempted from such registration) and have obtained a BSc degree or above in a relevant subject plus 5 years of post-Part I registration supervisory experience in constitutional cytogenetics, or have obtained a recognised overseas qualification in cytogenetics from a university and/or professional body.

3.5Both the medical personnel who provides clinical interpretation and the technical personnel involved in producing the test results for chromosomal microarray analysis (CMA) shall be familiar with the principles of the software programme being applied, and up-to-date information on the annotation and clinical significance of copy number variation (CNV).

4Laboratory equipment, reagents, and consumables

4.1Procedures to assure and verify the proper functioning of equipment shall meet acceptable standards, e.g.HOKLAS Supplementary Criteria No. 38 "'Medical Testing' Test Category - Performance Verification of Automated Analysers".

4.1.1Tissue culture incubators shall have a system to monitor the temperature continuously, and an alarm system to alert laboratory staff of abnormal culture conditions.

5Pre-examination processes

5.1Both the request form and specimen submitted for cytogenomic studies shall each contain at least two independent identifiers for unique identification of the patient. The identifying information on the request form shall be identical to that on the specimen tube label. There shall be a system to identify the person collecting the specimen for the test.

5.2The laboratory shall only accept test requests for CMA, where relevant, with appropriate genetic counselling and consent documented.

6Examination processes

6.1 Each prenatal specimen for cytogenetic studies shall be divided, cultured in two separate incubators and maintained with independent cell cultures, media and reagents. Duplicate or independently established cultures should be included if adequate specimenis available.

6.2For cytogenetic studies, adequate number of banded metaphases should be examined. In general, a minimum of 5 cells (10 for cancer cytogenetics) should be analysed, and at least two cells shall be checked by a qualifiedpersonnel as defined in clauses 3.1 to 3.4. When there is evidence of mosaicism or clonal evolution, further cells should be examined.

6.3For FISH studies, an effort should be made to examine a few metaphases with reverse DAPI chromosome staining to confirm that the correct probes have been used and to identify any unusual signal pattern. If metaphase is absent or inherent control signal is not available, the test should be repeated in parallel with another sample known to have the target of the probes.

6.3.1There shallbe a clear and consistent definition of fluorescence signals andalso criteria for false positive and false negative signals.

6.3.2 Adequate number of interphase nuclei or metaphases should be examined. In general, a minimum of 5 metaphases and/or 100 interphase nucleishould be studied and scored. FISH signals shall be scored independently by 2 people.

6.4 For CMA, the laboratory shall have written procedures for DNA extraction, quality, yield and quantitation, proper fragmentation and fluorescent labelling.

6.5CMA of cancers often requires the testing of small genomic aberrations in a mixed cell population.The laboratory shall understand the limitations of the test and in particular, the type of specimens (such as blood,fresh and formalin-fixed paraffin embedded tissues), tumour heterogeneity, contamination by normal DNA, selection of CMA platforms, coverage of the region of interest and the need forreference DNA (matched or pooled).

6.6For CMA, the followingexperiment design and validation shall be considered:

6.6.1All processes and parameters of CMA, including new array designs, shall be validated using DNA from a range of known abnormal samples, including numerical and structural variation, microdeletion and microduplication. Correct identification of a defined and reasonable number of abnormal specimens is suggested. Where alimited number is used for validation, there shall be documentation of justification and on-going data accumulation to complete the validation process. For validation of an updated version of a microarray platform, correct identification of at least five known abnormal samples is suggested.

6.6.2If an enhanced (or a new) version of platform is used, it shall be validated withabnormal samples that reflect the additional capacity of the platform. A single algorithm or software programme shall be used throughout.

6.6.3If an in-house assay is developed, the laboratory shall validate each clone or region using alternative method and reference DNA.

6.6.4For an individual application, a single algorithm and set of parameters shall be used for all data analysis. The laboratory shall re-analyse all data used for initial validation if an alternative algorithm is used.

6.6.5Software packages should produce diagrammatic and numerical output for analysis. Software parameters shall be defined and documented to ensure detection of imbalance at, or greater than, the level specified by the laboratory. A minimum genome-wide resolution at 400 kb is recommended.

7Ensuring quality of examination results

7.1For tests that give quantitative results, the laboratory shall determine the uncertainty of measurement and document the uncertainty components. An example is FISH study with quantitative results.

7.2All staff taking part in the testing activities shall participate inappropriate external quality assessment or interlaboratory comparison programme.

7.3In the absence of EQAP, both normal and abnormal samples shall be included in the interlaboratory comparison programme.

7.4The laboratory shall have a standard protocol for the confirmation of abnormal or ambiguous results for CMA by alternative techniques, such as FISH, PCR, as far as is reasonably practicable.

8Post-examination processes

8.1A minimum of two karyotypes/images shall be prepared and archived.

8.2Storage of the primary specimens and other laboratory samples shall be in accordance with the requirements given in Table 1. Laboratories should retain records and/or materials for a longer period of time than specified when such is appropriate for patient care, education, quality improvement needs or legal requirements, etc.

9Reporting of results

9.1The description of test results shall follow the latest version of the International System for Human CytogenomicNomenclature (ISCN).For cancer cytogenetics and FISH studies in haematology,the tests shall be reported by a qualified haematologist with appropriate training and laboratory experience.

9.2For FISH studies inAnatomical Pathology, the tests shall be reported by a qualified anatomical pathologist with appropriate training and laboratory experience.

9.3For constitutional cytogenetics, the testsshall be reported by a medically qualified person with appropriate training and laboratory experience as required in sections 3.1 to 3.3. For those tests with normal results,numerical autosomal or sex chromosome abnormalitiesof well-recognised syndromes and common chromosomal polymorphism (an indicative list is given in Appendix 1), they may be reported by a biomedical scientist. Clinical interpretation shall not be provided in such reports.Communication of the significance of the finding to referring doctor is advised.

9.4For CMA, the following information shall be provided in the report forthe diagnosis of constitutional disorders in addition to the information described in clause 5.8.3 of HOKLAS 015 (5th edition):

  1. Date of specimen reception at the laboratory;
  2. Pedigree information (optional);
  3. Clinical disorder or indications for testing;
  4. Gene(s) and/or specific region or mutation(s) of interest analysed, if applicable (standardnomenclature of Human Genome Organisation Gene Nomenclature Committee (HGNC) shall be used);
  5. Brief description of the technical information, such as commercial source, the number and type of clones and loci tested, the version number of array chip and software programme used for the analysis;
  6. Database accession number and version number for the reference sequence or genome, if applicable;
  7. Test performance specifications and limitations (e.g. mosaicism, balanced rearrangements, etc);
  8. Test sensitivity with quoted source reference, if available (e.g. the proportion of affected individuals likely to be detected);
  9. A remark to notify the requesting clinician that post-test genetic counselling service has to be provided to the patient if necessary;
  10. Comments or recommendations on family study;
  11. A summary statement, where applicable (e.g. current ISCN nomenclature for copy number variant identified).

9.5 The laboratory shall have established criteria to identify copy number variation (CNV) and the corresponding reportable range in CMA with reference to international guidelines such as the American College of Medical Genetics and Genomics (ACMG).

  1. Standard abnormality and mutation nomenclature (e.g. The Human Genome Variation Society (HGVS) nomenclature and An International System for Human Cytogenomic Nomenclature) shall be used for result reporting. Commonly used name for the abnormality and mutation may also be included in the report for further clarification where appropriate.
  2. When reporting negative test results, “no abnormality has been detected”shall be used instead of “normal”.
  3. Disclaimers such as the possibility of errors due to factors beyond the control of the laboratory (e.g. the risk of mistaken paternity and the knowledge of family relationship as stated), where appropriate, should be mentioned in the report.
  4. Interpretation of the residual risk remaining after a negative CMA result, when applicable, should be included in the report. This risk assessment is a quantitative statement of the predictive value of the result. It is usually based on the CNVfrequency and test sensitivity. Bayesian calculation can be used to estimate the residual risk.
  5. References should be given when published data have a bearing on the interpretation or risk calculation. In general, inclusion of references is only necessary when the data are not widely known or accepted. When different publications have shown conflicting results, it is important to specify which has been used as the basis for the interpretation.
  6. The laboratory should have a written policy on the reporting of absence of heterozygosity (AOH) for SNP array.

9.6The laboratory shall keep information of available genetic counselling service.

9.7Where appropriate, the test report shall include a recommendation that the patient should obtain genetic counselling from a qualified healthcare professional.

- End -

Table 1 Retention of Laboratory Records and Materials

Record/material / Requirement
General / Records of employee signatures, initials, and identification codes / 10 years
Referring doctor's request / 3 years after dispatch of final report.
Indefinite for request form which contains clinical information not readily accessible in the patient’s notes but used in the interpretation of test result.
Where the request form is used to record working notes or as a worksheet, it should be retained as part of the laboratory record.
Cytogenetics/Fluorescence in situ hybridisation/ Chromosomal microarray analysis / Copies of reports / Indefinite
Patient information/karyotypes / Indefinite
Slides / 3 years after final report if photographic record kept; 5 years otherwise, unless degeneration evident
Original specimen and container/wet specimen/tissue / 1 month after reporting
Fixed chromosome preparation (blood, bone marrow) / Abnormal: indefinite
Normal: 6 months
Tissue culture/cell culture / Cryopreserved, and indefinite when appropriate
Diagnostic images (digitised or negatives) / Indefinite
CMA data / Indefinite

N.B.: Indefinite means without limit of time, but not less than 30 years.

Appendix 1

An indicative list of the numerical autosomal or sex chromosome abnormalities of well-recognised syndromes and common chromosomal polymorphism.

A.Non-mosaic numerical abnormalities:

  1. 47,XX,+21 or 47,XY,+21
  2. 45,X
  3. 47,XX,+13 or 47,XY,+13
  4. 47,XX,+18 or 47,XY,+18
  5. 47,XXY
  6. 47,XXX
  7. 47,XYY
  8. 69,XXX or 69,XXY or 69,XYY

B.Common chromosomal polymorphism:

  1. 46,XX,inv(9)(p11q13) or 46,XY,inv(9)(p11q13)
  2. 46,XX,inv(9)(p11q12) or 46,XY,inv(9)(p11q12)

SC-35 Annex: Checklist on compliance with HOKLAS requirements–Cytogenomics Page 1 of 16

Issue No. 3

HOKLAS Requirement / Clause (HOKLAS 015, 5th edition and relevant SC) / *1 / Y / N / NA / Lab’s Document Reference or Remarks2 / Assessment Team’s remarks /
questions to be asked at the
laboratory
Please ensure that the General Checklist (HOKLAS 021) is also completed for each discipline.
Discipline Specific Management Requirements
Quality and technical records / 4.13
Do laboratory records indicate the media used, culture conditions and incubation times for all preparations? / 4.13 (e) / ●
Do laboratory records include the number of cells counted, analysed microscopically and the cells from which photographic or digitalised karyotypes are prepared? / 4.13 (h) / ●
Do laboratory records include an assessment of banding resolution to indicate whether metaphase analysis is satisfactory? / 4.13 (h) / ●
Discipline Specific Technical Requirements
Laboratory equipment, reagents, and consumables / 5.3
Does the laboratory have adequate number of image processing systems? / 5.3.1.1 / ●
Are microscopes equipped for high-resolution cytogenetics analysis? / 5.3.1.1 / ●
Are cell cultures manipulated under conditions that ensure sterility and protect staff? / 5.2.6 / ●
Is there a class II biosafety cabinet that is certified annually in the laboratory? / 5.3.1.5 / ●
Are incubators fitted with alarms or override systems that protect against malfunction of temperature and CO2 controls? / SC35 4.1 / ●
Is the quality of optical image-capture system high enough to minimise image degradation? / 5.3.1.2 / ●
Pre-examination processes / 5.4
Does the information provided on the request form include gender and date of birth which are important for interpretation of genetic testing results? / 5.4.3 (a) / ●
Are there procedures to verify sample identity and integrity? / 5.4.4.3 / ●
Are at least two unique identifiers provided in both the request forms and specimens submitted for cytogenomic studies? / SC35 5.1 / ●
Is there a system to ensure the person collecting the specimens for cytogenomics and FISH tests can be identified? / SC35 5.1 / ●
Examination processes / 5.5
Is each lot of culture medium checked for sterility? / 5.3.2.3 / ●
Are there independently established or duplicate cultures to backup against unexpected failures, unless the sample contains insufficient cells to do so? / SC35 6.1 / ●
Are at least two cells checked by a qualified personnel? / SC35 6.2 / ●
When there is evidence of mosaicism or clonal evolution, are further cells examined? / SC35 6.2 / ●
Constitutional Cytogenetics
Is prenatal specimen for cytogenetic studies divided, cultured in two separate incubators and maintained with independent cell cultures, media and reagents? / 5.5.1.1
SC35 6.1 / ●
Are duplicate prenatal culture flasks or dishes harvested independently? / 5.5.1.1 / ●
For prenatal cultures, are at least two independent cultures analysed? / 5.5.1.1 / ●
Are the band level, quality of banding and resolution sufficient to render the reported interpretation? / 5.5.1.1 / ●
Are at least 5 cells counted? / 5.5.1.1
SC35 6.2 / ●
Are at least 5 cells analysed? / SC35 6.2 / ●
Cancer Cytogenetics
Are culture methods, culture media and additives selected according to the nature of neoplastic disorder under study? / 5.5.1.1 / ●
Are at least 10 cells analysed, if possible? / 5.5.1.1
SC35 6.2 / ●
Are at least 2 karyotypes per mainline and 1 karyotype each from pertinent sidelines generated for each case? / 5.5.1.1 / ●
FISH analysis
Has the laboratory established the reference range(s) for each probe used? / 5.5.2 / ●
Are metaphases with DAPI chromosome staining to confirm that the correct probes have been used and identify any unusual signal pattern? / SC35 6.3 / ●
If metaphase is absent or inherent control signal is not available in FISH studies, is the test repeated in parallel with another sample know to have the probe target? / SC35 6.3 / ●
Are there a clear and consistent definition of fluorescence signals and also criteria for positive and false negative signals? / SC35 6.3.1 / ●
Are at least 5 metaphases and/or 100 interphase nuclei studied and scored? / SC35 6.3.2 / ●
Are FISH signals scored independently by 2 people? / SC35 6.3.2 / ●
Chromosome instability syndromes analysis
If breakage studies are performed, are they performed with concurrent controls in the same manner as patient specimen? / 5.6.2.2 / ●
Post-examination processes / 5.7
Are at least two karyotypes/images prepared and archived? / SC35 8.1 / ●
Are fixed preparations of chromosomes and cells stored indefinitely if abnormal, and for 6 months if normal? / SC35 Table 1 / ●
Are slides stored for 3 years after final report if photographic record kept, or 5 years otherwise, unless degeneration is evident? / SC35 Table 1 / ●
Are negatives, prints or retrievable electronic media stored indefinitely? / SC35 Table 1 / ●
Are copies of reports stored indefinitely? / SC35 Table 1 / ●
Is original specimen and container/wet specimen/tissue stored one month after reporting? / SC35 Table 1 / ●
Is harvested tissue culture/cell culture stored indefinitely when appropriate? / SC35 Table 1 / ●
Reporting of results / 5.8
Does the cytogenetics report contain:
-biological sex of the patient? / 5.8.3 / ●
-description of specimen / tissue studied? / 5.8.3 / ●
-number of cells (metaphases) analysed, counted and karyotyped? / 5.8.3 / ●
-banding method(s) used? / 5.8.3 / ●
-diagnosis and classification according to the WHO classification (for haematology malignancies)? / N/A / ●
Is the current International System for Human Cytogenomic Nomenclature (ISCN) used correctly in the final report for conventional cytogenetic studies? / SC35 9.1 / ●
Does the FISH report include information on the limitations of the test applied, on the source of the probe and on the implications of the result? / 5.8.1 / ●
Is the final report for constitutional cytogenomics reported by a medically qualified person with appropriate training and laboratory experience as required in SC35 Clauses 3.1 to 3.3? / SC35 9.3 / ●
Do all reports specified in Clause 9.3 reported by a biomedical scientist show the following mandatory remark: "Test results shown on this report require clinical interpretation and comments by a qualified pathologist (or equivalent as advised by the Hong Kong College of Pathologists)”? / SC35 9.3 / ●
Are appropriate genetics counselling referrals available for genetic testing and have these been documented in the laboratory’s procedures? / SC35 9.6 / ●

Note: 1. The assessor should concentrate on items marked with a ●; other items will be checked by the team leader.