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HelpTest Code LAB3822 SpotSeq ddPCR
Clinical System Name
SpotSeq
Synonyms
High Sensitivity Allele Specific Assay
Description
SpotSeq is a clinical-grade droplet digital PCR (ddPCR) assay for targeted mosaic variant testing on cfDNA, fresh/frozen tissue, blood, or saliva. Cell-free DNA can be derived from lymphatic cyst fluid or whole blood (plasma). Please note that FFPE specimens will not be accepted at this time.
SpotSeq offers targeted, high sensitivity variant detection with low input material. The Seattle Children's Hospital Molecular Laboratory offers different SpotSeq panels based on clinical indication.
Please select ONE target per request. Each target is a separate test and a separate charge.
Detailed descriptions are below.
|
Gene |
Variant(s) |
Target |
|
PIK3CA |
p.E542K, p.E545K, p.H1047R |
Multiplex; 3x Hotspot |
|
TEK |
c.2740C>T, p.L914F |
Singleplex |
|
GNAQ |
c.548G>A, p.R183Q |
Singleplex |
|
BRAF |
c.1799T>A, p.V600E |
Singleplex |
| ARPC4 | c.472C>T, p.R158C |
Singleplex |
|
At this time we cannot accept requests for custom alleles. |
||
Please contact Lab Client Services (206-987-2617 or labclientservices@seattlechildrens.org) with questions about sample requirements and pricing and our Laboratory Genetic Counselors (206-987-5400 or LabGC@seattlechildrens.org) with other questions about these tests.
No reflex option. The option to add on Vascular Anomalies Sequencing Panel (VANseq), a NGS test, may be available if there is sufficient gDNA remaining (VANseq is NOT validated for cfDNA at this time) but a new order needs to be placed for VANseq (TEST CODE: LAB1920).
Sample Requirements
Samples MUST have two of the following to be accepted as properly labeled: first & last name, outside medical record number, unique accession number, or date of birth.
- Streck Cell-Free DNA BCT® tubes are required for cell-free DNA (cfDNA) testing.
- Tissue sampled from an affected site is preferred over non-affected tissue.
- Please select one sample type for submission. Paired sample testing is NOT accepted unless approved by lab director.
- Non-validated sample types will not be accepted for testing. Lymphatic cyst fluid is the ONLY acceptable body fluid. We do not accept: pleural fluid, chylous fluid, pericardial fluid, peritoneal fluid or FFPE.
- For patients who have had a whole blood transfusion, wait 10 days post transfusion to draw blood or collect saliva for genetic testing. No wait time is necessary for blood or saliva collection if the patient received leuko-reduced red cells or plasma.
- Please contact LabGC@seattlechildrens.org if you would like assistance selecting the most appropriate sample type.
| Sample | Requirement | Important notes | |
| Blood (for cfDNA) | 16-20 mL whole blood in Streck Cell-Free DNA BCT® tube | Sample must be received within 5 days of collection, ship overnight at room temperature. Tubes cannot be frozen. | |
| Lymphatic cyst fluid | 1-8 mL in Streck Cell-Free DNA BCT® tube | Sample must be received within 5 days of collection, ship overnight at room temperature. Tubes cannot be frozen. | |
| Fresh frozen tissue (-70C) |
25-50 mg in a sterile container. |
A copy of the pathology report (if available) is recommended.
Specify tissue source and site on requisition. |
|
| Skin biopsy |
2-4 mm punch biopsy of skin collected under sterile conditions in a sterile vial, frozen with no media.
Also acceptable, refrigerate with 1-3 mL of tissue transport medium. |
DO NOT use formaldehyde, formalin, alcohol, or 5% dextrose.
Specify tissue source and site on requisition. |
|
| Extracted gDNA or cfDNA |
2-5 mcg DNA from tissue or EDTA blood
DNA concentration minimum 50 µg/mL (40uL minimum volume); 260/280 ratio 1.70-2.00.
cfDNA extracted externally may be permitted below these concentrations. Prior approval from the laboratory is required, please contact LabGC@seattlechildrens.org |
Isolation of nucleic acids for clinical testing must be performed in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or the CMS. | |
| Saliva |
Oragene Dx OGD-575/675 collection kit
Contact the lab directly for more information or to obtain a kit - 206-987-2563, ReferenceLabTeam@seattlechildrens.org. |
Manufacturer instructions must be followed. Please review link: How to Collect a Saliva Sample for Genetic Testing (Spanish). | |
| Blood (for gDNA) |
1-3 mL in Lavender/EDTA tube |
Rare specimen type for this assay. For the extraction of genomic (NOT cell-free) DNA. |
|
Processing Instructions
| Specimen Type | Description |
Temperature |
Storage instructions |
| Cell-free DNA, whole blood | Streck tube | Room Temp | Place in CPA Cytogenetics room temp box with requisition |
| Lymphatic cyst fluid | Streck tube | Room Temp | Place in CPA Cytogenetics room temp box with requisition |
| Extracted DNA | DNA aliquot tube | Refrigerate | Molecular Genetics box in CPA refrigerator #2 |
| Fresh frozen tissue | Frozen aliquot of 25-50 mg tissue | Frozen (-70C) | CPA -70 freezer (SNP array/Molecular box) |
| Skin biopsy | Sterile container (no media) | Frozen (-70C) | CPA -70 freezer (SNP array/Molecular box) |
| In medium | Refrigerate | Molecular Genetics box in CPA refrigerator #2 | |
| Saliva | OGD-575/675 kit | Room Temp | Place in CPA Cytogenetics room temp box with requisition |
| Whole blood (WBC) | EDTA or ACD tube | Refrigerate | Molecular Genetics box in CPA refrigerator #2 |
Off-site collection: Transport blood, lymphatic cyst fluid, or extracted DNA at room temperature via overnight shipping. Transport fresh frozen tissue on dry ice.
Stability
| Specimen Type | Temperature | Time |
|---|---|---|
| Cell-free DNA, whole blood | RT | 2 weeks |
| Lymphatic cyst fluid | RT | 2 weeks |
| Tissue - Fresh Frozen | -70 C | years |
| Whole blood (WBC) | RT | 5 d |
| Whole blood (WBC) | 2 - 8 C |
7 d |
|
Saliva fresh or extracted from ORAgene Dx OGD-575/675 |
refrigerated or room temp | up to 2 weeks |
| Extracted DNA | RT, refrig or frozen | stable |
Note: Whole blood samples >5 days may be submitted to be assessed by our lab for acceptability for testing.
Availability
| STAT | Performed | TAT |
|---|---|---|
| Contact lab | Monday - Friday | 21 days |
Performing Laboratory
Seattle Children's Laboratory
Department
Department: Molecular Genetics Laboratory, labdnabank@seattlechildrens.org
Phone: 206-987-3872
Lab Client Services: 206-987-2617 or labclientservices@seattlechildrens.org
Lab Genetic Counselor: LabGC@seattlechildrens.org
CPT Codes
| Gene | Variant | CPT | ||
| PIK3CA | c.1624G>A | p.E542K | p.Glu542Lys | 81309 |
| c.1633G>A | p.E545K | p.Glu545Lys | ||
| c.3140A>G | p.H1047R | p.His1047Arg | ||
| TEK | c.2740C>T | p.L914F | p.Leu914Phe | 81479 |
| GNAQ | c.548G>A | p.R183Q | p.Arg183Gln | 81403 |
| BRAF | c.1799T>A | p.V600E | p.Val600Glu | 81210 |
| ARPC4 | c.472C>T | p.R158C | p.Arg158Cys | 81479 |
Methodology
Method: Allele-specific droplet digital PCR (ddPCR) targeting single nucleotide variant(s). The limit of detection for this assay is typically 0.3% mutated alleles.
See Description above for reported variant set of each panel
Limitations:
Variants located outside of the targeted region(s) will not be detected. Samples with mutations below the limit of reporting may not be detected.
Reference Range
Results are reported as NEGATIVE if no variant is detected. Samples with a variant detected are reported POSITIVE and the specific variant is reported. Interpretive report will be provided.
Clinical Utility
- Testing cell-free DNA or tissue for a suspected mosaic disorder
- Patients who are positive for a PIK3CA mutation may be eligible for targeted therapies
- Clinical confirmation of a suspected mosaic finding identified by a research study