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Test Code LAB3822 SpotSeq ddPCR

Important Note

Please note the specimen requirement of blood for cell-free DNA testing has INCREASED to 16-20mL per test.  For blood collection only, two tubes are required for each gene selected. 

Required sample volumes for other specimen types remain unchanged.

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.

 

Given the distinctive phenotypes associated with each of these genes and the targeted nature of this testing, we do not recommend completing multiple SpotSeq tests concurrently. Each target is a separate test and a separate charge. This SpotSeq testing is most informative when you already have a strong suspicion for one of these target genes, and would like to confirm a molecular diagnosis despite lack of available tissue. If there is still uncertainty around possible diagnoses, broader testing is likely to be more informative, please see Vascular Anomalies Sequencing Panel (VANseq) for other options.

 

Gene

Targeted Variant(s)

Clinical Phenotype

PIK3CA

p.E542K, p.E545K, p.H1047R

80% of isolated lymphatic malformations have pathogenic, tissue/lesion restricted variant in PIK3CA1. PIK3CA SpotSeq using lymphatic cyst fluid is likely to be our highest yield offering.

TEK

c.2740C>T, p.L914F

Somatic activating variants in TEK are the most common cause of sporadically occurring unifocal venous malformations2. Activating TEK variants can also cause multifocal venous malformations and blue rubber bleb nevus syndrome.

GNAQ

c.548G>A, p.R183Q

This variant accounts for ~90% of individuals with congenital isolated capillary malformations (MIM: 163000) and Sturge-Weber syndrome (MIM: 185300)3. This variant has also been reported in phakomatosis pigmentovascularis and extensive dermal melanocytosis4. 

BRAF

c.1799T>A, p.V600E

Activating, somatic mutations in BRAF can be associated with a wide variety of vascular and non-vascular cutaneous tumors, such as arteriovenous malformations, isolated lymphatic malformations, pyogenic granuloma, wooly hair nevus, melanocytic nevi, and sebaceous nevus5.

 

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.  Please be aware that VANseq is NOT validated for cfDNA at this time. A separate request must be submitted, please contact lab to discuss. 

 

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.

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
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.
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.

Fresh tissue or fresh frozen tissue (-70C)

 

25-50 mg in a sterile container - (cryovial preferred).

 

OR

 

2-4 mm punch biopsy of skin collected under sterile conditions in a sterile vial, frozen with no media.  

 

Please refrain from sending specimens weighing greater than 1 gram.

Specify tissue source and site on requisition.

 

A copy of the pathology report is required.

 

Preferred storage/transport conditions: frozen -70C

 

Also acceptable: refrigerate with 1-3mL of tissue transport medium (eg. RPMI with fetal calf serum & antibiotics, or Fibroblast Transport Media, or Hank's Balanced Salt Solution or sterile saline). 

 

NOT ACCEPTABLE: formaldehyde, formalin, alcohol, or 5% dextrose.  
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
Lymphatic cyst fluid Streck tube Room Temp Place in CPA Cytogenetics room temp box with requisition
Cell-free DNA, whole blood 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

Requisition

Molecular VANseq & SpotSeq Requisition

References

  1. Zenner et al. 2019, Luks et al. 2015
  2. Ye et al., 2011, Soblet et al., 2017, Seront et al., 2023
  3. Shirley et al., 2013, Nakashima et al., 2014, Frigerio et al., 2015, Couto et al., 2016, Siegal et al., 2018, Bichsel et al., 2019
  4. Thomas et al., 2016, , Kumar et al. 2019
  5. Al-Olabi et al., 2018, Zenner et al., 2022, Salgado et al., 2015, Groesser et al., 2016, Ponti et al., 2007

 

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