PRENATAL TESTING
Testing on prenatally collected samples of ongoing pregnancies. See information on specimen requirements and billing below.
Chromosomal microarray (CMA) is a molecular cytogenomic test for detecting both copy number changes (deletions and duplications) and copy-neutral absence of heterozygosity (AOH).
Indications for Prenatal CMA
- Abnormal ultrasound: structural anomalies or markers
- Abnormal NIPS result confirmation
- Family history of chromosome abnormality or copy number variant
Test Uses
Chromosomal microarray can be used for detection of the following genomic changes:
- Copy Number Variants (CNV) – duplications or deletions of chromosomal material
- Pathogenic and Likely Pathogenic CNVs are always reported
- Variants of Uncertain Significance (VUS) are reported if they meet reportable criteria
- Benign and Likely Benign CNVs are not reported
- Absence of Heterozygosity (AOH) – areas of identical chromosomal material
- AOH is not diagnostic but may narrow down areas and/or autosomal recessive genes of interest to direct further testing
- AOH of 5% or more of the autosomal genome will be reported due to the increased likelihood of shared ancestry/identity by descent. Close consanguinity is predicted when 10% or more of the autosomal genome is identical
- Uniparental Isodisomy is detectable by CMA while heterodisomy is not. Additional testing for imprinted disorders may be recommended
- Mosaicism of a second cell population at greater than ~20%
- CNV classification is determined according to Riggs, et al. (2020).
Follow-Up Testing
CGL often recommends follow-up studies to further elucidate positive CMA results. Recommendations, if any, appear in the proband’s result report. Follow-up testing may include:
- proband and/or parental karyotype to evaluate for a chromosome rearrangement
- parental and/or family member CMA testing for the CNV detected
- When a VUS is detected, two close biological family members can be tested by CMA without additional charge if relevant to patient care
- proband molecular, methylation, or biochemical testing of a gene or region of interest at an outside laboratory
Limitations
CMA will NOT detect balanced rearrangements (i.e. inversions, translocations, etc.), heterodisomy, or reliably identify low-level mosaicism (below ~20%). For mosaic conditions, CMA cannot differentiate between specific number of copies present (e.g. trisomy of a whole chromosome arm vs. tetrasomy in 50% of cells tested). It also does not detect deletions and duplications with limited probe coverage, gene expansions, variants within mitochondrial DNA, or single nucleotide variants/polymorphisms. CNVs that are smaller than the resolution of the CMA may not be detected or reported. CNVs determined to be benign or likely benign are not reported. Typically, carrier status is not reported unless possibly relevant to the patient’s reported symptoms to guide further testing. Contact the laboratory if there is a specific region or gene(s) of interest.
CMA may reveal whole genome mosaicism suggestive of the presence of two different genomes, as in the case of maternal cell contamination (MCC), which may limit the interpretation of CMA results. For products of conception, placenta, or chorionic villi samples, when a single female genome is detected, it is assumed to represent the female fetus. However, the rare possibility that the specimen analyzed is maternal in origin cannot be excluded; MCC testing (see below) is required to rule out this possibility. For multifetal pregnancies, it is assumed that each specimen received was sampled from a different placenta/amniotic sac.
Reporting Thresholds
| Finding | Reportable Size* |
|---|---|
| Loss/Deletion | 1 Mb or larger |
| Gain/Duplication | 2 Mb or larger |
| Absence of Heterozygosity | 10 Mb or larger always reported 3 Mb and larger are analyzed and may be reported if the total AOH is 5% or greater |
*Smaller AOH or copy number variants may be reported if likely to have relevant clinical significance
Methods
Extracted DNA is processed on the Illumina® Infinium Global Diversity bead array. The Infinium Global Diversity bead array targets approximately 4885 genes in accordance with the most recent ICCG recommendations. The Infinium Global Diversity bead array is scanned on Illumina’s iScan Platform. Using single nucleotide polymorphism (SNP) probes, the array can detect genomic copy number losses and gains, known as copy number variants (CNVs), with a resolution of approximately 5 kb across the genome. NxClinical software (Bionano Inc.) is used to analyze and interpret the data. CNVs devoid of relevant gene content or that are common CNVs in the general population (listed in the Database of Genomic Variants) may not be reported. Due to probe placement on the chromosomal microarray (CMA), the smallest size CNV that is detectable is variable by region.
Genomic linear positions are given relative to the Genome Reference Consortium Human Genome build 37 (hg19) released by the National Center for Biotechnology Information (NCBI). CNVs are analyzed using publicly available databases and literature. See the following references regarding CMA analysis, interpretation, and reporting: PMID: 31690835, 34131312, 34906464, 27875474.
Turn Around Time
7-14 days, though may be longer if cell culture is required
Chromosome analysis is performed on G-banded metaphase chromosomes for the purpose of detecting numerical and/or structural abnormalities. These include aneuploidy (such as trisomy or monosomy), mosaicism, unbalanced rearrangements such as interstitial or terminal deletions/duplications and/or balanced rearrangements such as translocations and inversions.
Indications
Standard (Routine) Chromosome Analysis
- Increased risk of aneuploidy due to advanced maternal age or abnormal screening
- Congenital anomalies on ultrasound
- Intrauterine growth restriction
- Family history of chromosome abnormality
5-cell Chromosome Analysis
- As a supplement to Chromosomal microarray (CMA) studies, 5 cells are evaluated to identify or rule out balanced chromosome rearrangements for the indications above. This is not a stand-alone test.
Methods
Metaphase chromosomes are prepared using methanol/acetic acid fixation and G-banding by trypsin-Leishman (GTL). The number of metaphases analyzed, counted, and karyotyped are enumerated in the patient’s report. Nomenclature is according to the current ISCN (2024). Conventional chromosome analysis cannot reliably detect submicroscopic copy-number changes (below 5-10Mb), cryptic rearrangements, or low-level mosaicism. The detection of abnormalities is limited by banding resolution and the presence of viable, dividing cells. If performed, five-cell chromosome analysis may not detect mosaicism. If mosaicism for aneuploidy is detected, additional cells may be counted.
CVS: For standard chromosome analysis, 20 cells are counted with 5 cells analyzed for chromosome number and structure at the 400 band level of resolution.
Amnio: For standard chromosome analysis, 15 cells are counted from 15 colonies with 5 cells analyzed for chromosome structure at the 400 band level of resolution. If fewer than 15 colonies are available, 20 cells are counted.
Limitations
Subtle chromosome rearrangements such as duplications or deletions smaller than 5-10 Mb or cryptic translocations are not detectable by chromosome analysis. Chromosome analysis cannot detect Uniparental Disomy, absence of heterozygosity (AOH), or sequence variants.
When an XX chromosome complement is detected, it is assumed to represent the female fetus. However, the possibility that the specimen analyzed is maternal in origin cannot be excluded; MCC testing is required to rule out this possibility.
Follow-up Testing
Some chromosome abnormalities may warrant additional testing by microarray including unbalanced rearrangements, apparently balanced rearrangements in an individual with symptoms, and copy number variants to determine the extent of chromosome material involved and identify affected genes. Chromosome analysis testing for family members may also be recommended when a chromosome rearrangement is identified.
Turn Around Time
10-14 days
Fluorescence in situ hybridization (FISH) detects chromosome aneuploidy and submicroscopic genomic copy number changes such as deletions and duplications in specific regions.
Indications
- Suspicion of aneuploidy (X, Y, 13, 18, 21)
- Sex chromosome evaluation
- Xcen/Ycen, Xcen/STS, Xcen/SRY as relevant
- Suspicion for specific disorders:
- Williams Syndrome/ELN (7q11.2)
- Prader-Willi/Angelman Syndrome (15q11.2)
- 22q11.2 Microdeletion Syndrome
- X-linked Ichthyosis Xp22.21
- Familial testing after diagnosis of proband
Limitations
FISH is not considered a diagnostic test, but may guide the next steps of testing. FISH probes bind to a specific loci in the genome and can provide a rapid, preliminary copy number assessment of an area of interest. However, several factors may lead to inaccurate FISH results. Co-localized signals, interrupted binding sites, very small copy number variants, and other events contribute to occasional false positives and false negatives.
As FISH cannot determine the extent of the genetic material duplicated or deleted, abnormal FISH results require follow-up with CMA and/or chromosome analysis to further clarify the scope of the genomic imbalance and its medical significance. Critical and irreversible medical decisions based on FISH results alone, such as termination of pregnancy are NOT recommended.
Occasionally, FISH may be recommended to clarify the results of chromosome analysis or CMA. Such additional recommendations will be discussed with the ordering provider prior to being performed.
When XX FISH signals are detected, the results are assumed to represent the female fetus. However, the possibility that the specimen analyzed is maternal in origin cannot be excluded; MCC testing is required to rule out this possibility.
Turn Around Time
1-3 day
Various methods of prenatal specimen collection pose an increased risk for maternal cell contamination (MCC) including chorionic villi samples (CVS), products of conception, and other placental specimens. Such contamination can result in ambiguous or even unusable results if not clarified. When not excluded by other test results, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) recommend that MCC testing be performed to optimize interpretation of the primary molecular and/or cytogenetic results (Nagan, et al. 2011).
Potential Indications
- Chorionic villi sampling
- Bloody amniocentesis specimens
- Placental or early POC specimens
Methods
A blood or buccal specimen from the person carrying the pregnancy is required for MCC testing and may be submitted along with a prenatal specimen.
MCC testing by short tandem repeat (STR) analysis is performed by Colorado Molecular Correlates Laboratory (CMOCO), a partner laboratory of Colorado Genetics Laboratory (CGL).
MCC is performed when requested depending on the specimen provided. MCC testing is typically performed concurrently with CMA, and as a reflex analysis if XX or mosaic XX results are found in the proband specimen. The decision of when and if to run MCC analysis is made in conjunction with the ordering provider.
For additional information, contact CGL Client Services at (303)724-5701.
Amniotic fluid alpha-fetoprotein (AF-AFP) testing at 15-30 weeks gestation screens a fetus for an increased risk of open neural tube or open body wall defects. AF-AFP analysis can be used to further evaluate abnormal serum AFP levels identified during maternal serum screening. Acetylcholinesterase (AChE) testing is performed as a reflex confirmation test on individuals with an abnormal amniotic fluid alpha-fetoprotein (AF-AFP) level of 2.0 multiples of the mean (MOM) or greater.
Indications
- Increased risk for neural tube defects by ultrasound or serum screening
- Increased risk for body wall defects by ultrasound or serum screening
- Routine rule-out of neural tube or body wall defects when other prenatal screening is ordered
Methods
AF-AFP testing is performed by the Associated Regional University Pathologists, Inc. (ARUP). Results and interpretation of the AChE are provided to CGL by ARUP.
Limitations
afAFP and AChE testing cannot be performed on a chorionic villi sample and requires an amniotic fluid specimen between 15-30 weeks gestation. Positive/abnormal results from this testing are not specific to a certain genetic or physical diagnosis but contribute to the overall clinical likelihood of a disorder.
Turn Around Time
Up to 14 days
Cryopreservation
Colorado Genetics Laboratory can culture and cryopreserve specimens for possible future studies. Specimens are typically held for two years but can be held longer at the provider’s request. Additional fees may be applied for additional storage time.
If testing on cryopreserved cells is desired, CGL will thaw and regrow the cells. Cultured cells can then be tested or sent to the desired reference laboratory for biochemical or molecular testing.
Send-out Testing
Colorado Genetics Laboratory can send extracted DNA, uncultured CVS cells or amniocytes, or cultured cells to other laboratories for additional molecular or biochemical studies upon request.
To coordinate send-out, the following items MUST be provided before a specimen can be shipped:
- Provide reference laboratory information and shipping details on the test requisition form
- Provide a FedEx shipping number or email a prepaid FedEx shipping label to client services at [email protected]
- Send a completed copy of the reference laboratory's test requisition form to CGL to include with the specimen
Please contact the lab when tissue culture for molecular or biochemical testing is needed BEFORE the specimen is obtained to help coordinate this testing.
If a specimen is not of adequate quantity for all testing orders and send out, CGL will contact the ordering provider for a priority of testing. If desired, CGL will culture cells prior to send out and maintain a portion of the cell culture for one week after send-out. Longer-term storage is available with cryopreservation.
REFERENCES
Committee on Genetics and the Society for Maternal-Fetal Medicine. Committee Opinion No.682: Microarrays and Next-Generation Sequencing Technology: The Use of Advanced Genetic Diagnostic Tools in Obstetrics and Gynecology. Obstet Gynecol. 2016 Dec;128(6):e262-e268. PMID: 27875474.
Gonzales PR, et al. Interpretation and reporting of large regions of homozygosity and suspected consanguinity/uniparental disomy, 2021 revision: A technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2022 Feb;24(2):255-261. Epub 2021 Dec 3. PMID: 34906464.
Nagan N, et al. Laboratory guidelines for detection, interpretation, and reporting of maternal cell contamination in prenatal analyses a report of the association for molecular pathology. J Mol Diagn. 2011 Jan;13(1):7-11. Epub 2010 Dec 23. PMID: 21227389; PMCID: PMC3069929.
Riggs ER, et al. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Med. 2020 Feb;22(2):245-257. Epub 2019 Nov 6. Erratum in: Genet Med. 2021 Nov;23(11):2230. PMID: 31690835; PMCID: PMC7313390.
Shao L, et al. Chromosomal microarray analysis, including constitutional and neoplastic disease applications, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2021 Oct;23(10):1818-1829. Epub 2021 Jun 15. PMID: 34131312.
Wapner RJ, et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. N Engl J Med. 2012 Dec 6;367(23):2175-84. PMID: 23215555; PMCID: PMC3549418.