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Genomic Sciences and Precision Medicine Center (GSPMC)

Laboratory Services

Testing

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Single Site Variant Sequencing (CCS PRO or CCS KFV)

Test Name
Single Site Variant Sequencing (CCS PRO or CCS KFV)

CPT Code
Gene/Variant Dependent or 81479

Test Information/Description
Custom clinical sequencing (CCS) is used to confirm specific variants of interest in the proband or in family members interest.

Methodology
When a variant of interest is identified, the DNA sequence of a segment of the genome surrounding each variant is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy (Sanger) sequencing.

Test Limitations
Certain types of sequence variation are difficult to identify and have not been validated to be reliably detected for current clinical use. These include insertions, deletions, copy number variations, triplet repeat expansions, and structural chromosomal arrangements.

Specimen Type

  1. Whole Blood – 2-4mL of whole blood in a purple top EDTA tube (2 tubes per patient is preferred). For infants, we require a minimum of 1mL of blood. The tube(s) must be labeled with at least two patient identifiers. Ship blood tube(s) overnight at room temperature in an insulated container within five (5) days of collection.
  2. gDNA – 10µg of purified gDNA with a minimum of 75ng/µL and a 260/280 purity ratio of 1.75-2.0 in a screw cap tube. The tube must be labeled with at least two patient identifiers. Ship gDNA overnight at room temperature. We do not accept products of genome amplification or other amplification reactions. DNA must be extracted in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.
  3. Buccal/Saliva – We accept buccal swabs and saliva samples collected in Oragene◦DxDNA® collection kits following the manufacturer's instructions.
  4. Fresh Frozen Tissue Specimens – 50mg of fresh tissue frozen within minutes after collection and stored at -80 degrees C. The specimen must be labeled with at least two patient identifiers. Ship tissue specimen(s) frozen on dry ice to arrive Monday-Friday.
  5. Fresh (Unfixed Tissue Slides – At least two (2) slides frozen within minutes after collection and stored at -80 degrees C. The specimen must be labeled with at least two patient identifiers. Ship tissue specimen(s) frozen on dry ice to arrive Monday-Friday. Due to the low yield of DNA derived from slides, testing for no more than two (2) variants is recommended.

Shipping Conditions
See above

Turnaround Time
30 Days

WES Trio Rapid

Test Name
WES Trio Rapid

CPT Code
WES Proband 81415, WES family member 81416

Test Information/Description
Whole Exome Sequencing (WES) is used to detect variants in a patient’s exome in order to determine the role of genomic variants in disease outcomes. The exome is a little more than 1% of the genome that codes for protein. The patient’s exome will be sequenced to an average depth of 100X with a minimum dept of coverage of 85X. Over 97% of the exome will be sequenced to a depth of 10X. The mitochondrial genome of the patient will be sequenced to a minimum depth of 20X.

Methodology
DNA will be extracted. The extracted DNA is quantified and sheared to the correct size. The sample then undergoes library preparation and the exome is captured using the Agilent SureSelect platform. After quality assurance, the captured library is then subjected to next generation DNA sequencing using the Illumina sequencing platform. The sequence variants are then loaded into a tertiary analysis software package that contains data sources and algorithms allowing for the evaluation of whole exome sequencing variants for evolutionary conservation, predicted impact on protein structure and function, ability to disrupt conserved splice sites, and presence in databases including OMIM, dbSNP, and HGMD. A custom software package is also used to determine what parts of the exome are not sequenced. When a variant of interest is identified, the DNA sequence of a segment of the genome surrounding each variant is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy (Sanger) sequencing. If parental/familial samples are submitted concurrently with the proband, segregation of alleles will be determined where this will enhance the interpretation of results.

Test Limitations
Using current sequencing and alignment technology, it is only possible to sequence 90-95% of the human reference exome to a minimum 10X coverage estimated to be required to reliably detect heterozygous variants. In addition, certain types of sequence variation are difficult to identify and have not been validated to be reliably detected for current clinical use. These include insertions, deletions, copy number variations, triplet repeat expansions, and structural chromosomal arrangements.

Specimen Type
Whole blood in EDTA tube, Saliva/Buccal swab collected in Oragene kit, gDNA extracted in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.

Specimen Requirements

  1. Whole Blood – 2-4mL of whole blood in a purple tope EDTA tube (2 tubes per patient is preferred). For infants, we require a minimum of 1mL of blood. The tube(s) must be labeled with at least two patient identifiers. Ship blood tube(s) overnight at room temperature in an insulated container within 5 days of collection.
  2. gDNA – 10µg of purified gDNA with a minimum of 75ng/µL and a 260/280 purity ration of 1.75-2.0 in a screw cap tube. The tube must be labeled with at least two patient identifiers. gDNA must be extracted in a CLIA or CMS equivalent laboratory. Ship gDNA at room temperature overnight. We do not accept products of genome amplification or other amplification reactions.
  3. Saliva/Buccal swab – Sample must be collected in Oragene collection kits. Ship kits overnight at room temperature.

Shipping Conditions
See above

Turnaround Time
30 days

WES Single or Multi-Sample (DUO, TRio, Quad, etc)

Test Name
WES Single or Multi-Sample (Duo, Trio, Quad, etc.)

CPT Code
WES Proband = 81415; WES family member = 81416

Test Information/Description
Whole Exome Sequencing (WES) is used to detect variants in a patient’s exome in order to determine the role of genomic variants in disease outcomes. The exome is a little more than 1% of the genome that codes for protein. The patient’s exome will be sequenced to an average depth of 100X with a minimum dept of coverage of 85X. Over 97% of the exome will be sequenced to a depth of 10X. The mitochondrial genome of the patient will be sequenced to a minimum depth of 20X.

Methodology
DNA will be extracted. The extracted DNA is quantified and sheared to the correct size. The sample then undergoes library preparation and the exome is captured using the Agilent SureSelect platform. After quality assurance, the captured library is then subjected to next generation DNA sequencing using the Illumina sequencing platform. The sequence variants are then loaded into a tertiary analysis software package that contains data sources and algorithms allowing for the evaluation of whole exome sequencing variants for evolutionary conservation, predicted impact on protein structure and function, ability to disrupt conserved splice sites, and presence in databases including OMIM, dbSNP, and HGMD. A custom software package is also used to determine what parts of the exome are not sequenced. When a variant of interest id identified, the DNA sequence of a segment of the genome surrounding each variant is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy (Sanger) sequencing. If parental/familial samples are submitted concurrently with the proband, segregation of alleles will be determined where this will enhance the interpretation of results.

Test Limitations
Using current sequencing and alignment technology, it is only possible to sequence 90-95% of the human reference exome to a minimum 10X coverage estimated to be required to reliably detect heterozygous variants. In addition, certain types of sequence variation are difficult to identify and have not been validated to be reliably detected for current clinical use. These include insertions, deletions, copy number variations, triplet repeat expansions, and structural chromosomal arrangements.

Specimen Type
Whole blood in EDTA tube, Saliva/Buccal swab collected in Oragene kit, gDNA extracted in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.

Specimen Requirements

  1. Whole Blood – 2-4mL of whole blood in a purple tope EDTA tube (2 tubes per patient is preferred). For infants, we require a minimum of 1mL of blood. The tube(s) must be labeled with at least two patient identifiers. Ship blood tube(s) overnight at room temperature in an insulated container within 5 days of collection.
  2. gDNA – 10µg of purified gDNA with a minimum of 75ng/µL and a 260/280 purity ration of 1.75-2.0 in a screw cap tube. The tube must be labeled with at least two patient identifiers. gDNA must be extracted in a CLIA or CMS equivalent laboratory. Ship gDNA at room temperature overnight. We do not accept products of genome amplification or other amplification reactions. DNA must be extracted in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.
  3. Saliva/Buccal swab – Sample must be collected in Oragene collection kits. Ship kits overnight at room temperature.

Shipping Conditions
See above

Turnaround Time
60 days

Whole Genome Sequencing Single and Multi-Family (Duo, Trio, Quad, etc)

Test Name
Whole Genome Sequencing Single and Multi-Family (Duo, Trio, Quad, etc.)

CPT Code
81425 (Proband); 81426 (family member)

Test Information/Description
Whole Genome Sequencing (WGS) is used to detect variants in a patient's genome in order to determine the role of genomic variants in disease outcomes. The patient's genome will be sequenced to an average depth of 40X with a minimum depth of coverage of 35X. Over 90% of the genome will be sequenced to a depth of 10X. The mitochondrial genome of the patient will be sequenced to a minimum depth of 20X.

Methodology
DNA will be extracted from whole blood. Extracted DNA is quantified and sheared to the correct size. The sample undergoes robotic library preparation followed by library QC. The prepared library is then subjected to next generation DNA sequencing on the Illumina platform. The reads from this sequencing are aligned to a reference sequence and variations from this reference are identified. The sequence variants are then loaded into a tertiary analysis software package that contains data sources and algorithms allowing for the evaluation of whole genome sequencing variants for evolutionary conservation, predicted impact on protein structure and function, ability to disrupt conserved splice sites, and presence in databases including OMIM, dbSNP, and HGMD. The software package annotates variants with this data, taking into account both the reference gene model and any identified alternate transcripts. A custom software package is used to determine what parts of the genome are not sequenced. When a variant of interest is identified, the DNA sequence of a segment of the genome surrounding each variant is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy (Sanger) sequencing or an alternate orthologous technique in the proband. If parental/familial samples are submitted concurrently with the proband, segregation of alleles will be determined where this will enhance the interpretation of the test results.

Test Limitations
Using current sequencing and alignment technology, it is only possible to sequence 90-95% of the human reference genome to the minimum 10-fold coverage estimated to be required to reliably detect heterozygous variants. In addition, certain types of sequence variation are difficult to identify and have not been validated to be reliably detected for current clinical use. These include insertions, deletions, copy number variations, triplet repeat expansions, and structural chromosomal rearrangements.

Specimen Type
Whole blood in ESTA tube or gDNA extracted in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or DMS.

Specimen Requirements

  1. Whole Blood – 2-4mL of whole blood in a purple top EDTA tube (2 tubes per patient is preferred). For infants, we require a minimum of 1mL of blood. The tube(s) must be labeled with at least two patient identifiers . Ship blood tube(s) overnight at room temperature in an insulated container within 5 days of collection.
  2. gDNA – 10µg of purified gDNA with a minimum of 75ng/µL and a 260/280 purity ratio of 1.75-2.0 in a screw cap tube. The tube must be labeled with at least two patient identifiers. Ship gDNA overnight at room temperature. We do not accept products of genome amplification or other amplification reactions.

Shipping Conditions
See above

Turnaround Time
60 Days

ATRX-IHC (Technical Component Only)

Test Name
ATRX-IHC (Technical Component Only)

CPT Codes
88342-TC, primary
88341-TC, if additional IHC

Indications for Ordering
Distinguishing differentiated from undifferentiated neoplasms.

Methodology
This test is intended to identify the presence of ATP-dependent helicase ATRX, X-linked helicase II (ATRX) protein. ATRX is produced by most mitotically active normal cells and can be useful in the distinction of differentiated from undifferentiated neoplasms.

Specimen Types

  1. Preferred: FFPE Tissue Blocks.
  2. (2) unstained positively charged glass slide (25- x 75- x 1-mm) per test ordered; sections 4-microns thick.

Specimen Stability
Ambient (preferred)

Results
This test includes only technical performance of the stain. The interpretation of the result will be provided by MCW Pathology department.

Test Limitations
No specific limitations for this test.

Turnaround Time
1-3 business days

Test Disclaimer
This test was developed, and its performance characteristics determined by Medical College of Wisconsin in a manner consistent with CLIA/CAP requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA).

Mismatch Repair (MMR) Protein Immunohistochemistry Only, Tumor

Test Name
Mismatch Repair (MMR) Protein Immunohistochemistry Only, Tumor

CPT Codes
88341 MLH-1, MSH-2, and MSH-6 Immunostain
88342 PMS-2, Immunostain

Indications for Ordering
Evaluation of tumor tissue to identify patients at risk for having hereditary nonpolyposis colon cancer/Lynch syndrome.

Methodology
Immunohistochemistry staining is used to determine the presence or absence of protein expression for MLH1, MSH2, MSH6, and PMS2. Lymphocytes and normal epithelium exhibit strong nuclear staining and serve as positive internal controls for staining of these proteins.

When this test is ordered, MLH1, MSH2, MSH6, and PMS2 stains will always be performed at an additional charge.

Specimen Types

Tumor tissue is required.

Preferred: FFPE Tissue Blocks

  1. Tissue Slides: 1 stained and 10 unstained
  2. (Collection Instructions: Submit 1 slide stained with hematoxylin and eosin and 10 unstained, non-baked slides with 4-micron thick sections of the tumor tissue).

Specimen Stability
Ambient (preferred)

Results
An interpretive report will be provided.

Test Limitations

  • The finding of absent protein expression for 1 or more of the MMR genes tested does not distinguish between somatic and germline mutations.
  • Because immunohistochemistry (IHC) results may indicate likelihood of a germline alteration, it is recommended that genetic counseling be provided prior to IHC testing.
  • Test results should be interpreted in the context of clinical findings, family history, and other laboratory data. Errors may occur in our interpretation of results if information given to us is inaccurate or incomplete.

Turnaround Time
7 business days

Test Disclaimer
This test was developed, and its performance characteristics determined by Medical College of Wisconsin in a manner consistent with CLIA/CAP requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA).

IDH1 Mutation (R132H) Immunostain (Technical Component Only)

Test Name
IDH1 Mutation (R132H) Immunostain (Technical Component Only)

CPT Codes
88342-TC, primary
88341-TC, if additional IHC

Indications for Ordering
IDH1 R132H can be used as a diagnostic marker to help differentiate infiltrating gliomas from gliosis, and as a prognostic marker for gliomas and secondary glioblastoma multiforme.

Methodology
Antihuman isocitrate dehydrogenase 1 (IDH1) R132H antibody binds to IDH1-mutated protein but does not bind the wild-type IDH1 protein. IDH1 R132H point mutations are frequently seen in World Health Organization grade II and III gliomas and are believed to constitute an early step in tumorigenesis. IDH1 R132H antibody shows strong cytoplasmic staining and weaker nuclear staining in tumor cells with the R132H-mutated peptide. Diffuse staining of the fibrillary tumor matrix is also seen.

Specimen Types

Preferred: FFPE Tissue Blocks.

  1. (2) unstained positively charged glass slide (25- x 75- x 1-mm) per test ordered; sections 4-microns thick.

Specimen Stability
Ambient (preferred)

Results
This test includes only technical performance of the stain. The interpretation of the result will be provided by MCW Pathology department.

Test Limitations
No specific limitations for this test.

Turnaround Time
1-3 business days

Test Disclaimer
This test was developed, and its performance characteristics determined by Medical College of Wisconsin in a manner consistent with CLIA/CAP requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA


PDL1 By Immunohistochemistry (Technical Only)

Test Name
PDL1 by Immunohistochemistry (Technical Only)

CPT Codes
88360

Indications for Ordering
Identification of neoplasms expressing programmed cell death 1-ligand 1.

Methodology
Programmed cell death 1-ligand 1 (PD-L1), also known as B7 homolog 1 (B7-H1) or CD274, is a transmembrane protein involved in the regulation of cell-mediated immune responses through interaction with the receptor programmed death protein-1 (PD-1). PD-L1 has been identified as both a prognostic and theranostic marker in a variety of neoplasms. Overexpression of PD-L1 has been observed in carcinomas of the urinary bladder, lung, thymus, colon, pancreas, ovary, breast, kidney, and in melanoma and glioblastoma.

Specimen Types

A pathology/diagnostic report and a brief history are required.

Preferred: FFPE Tissue Blocks.

  1. Air dried (with a specified cut date), 3 unstained slides (Dako FLEX IHC Microscope or Fisherbrand Superfrost Plus); sections 4-microns thick.

Specimen Stability
Ambient (preferred)

Results
The report will be provided by the MCW Pathology department.

Test Limitations
The result of this assay should not be used as the sole factor in determining treatment, as other factors (eg, tumor mutation burden and microsatellite instability) have also been studied as predictive markers.

This test has been validated for nondecalcified paraffin-embedded tissue specimens fixed in 10% neutral-buffered formalin. This assay has not been validated on tissues subjected to the decalcification process or use of alternative fixatives for bone and bone marrow specimens or cell blocks.

Turnaround Time
1-3 business days

Test Disclaimer
This test is approved by the U.S. Food and Drug Administration (FDA).

BRAF Mutation Analysis by Real Time PCR

Test Name
BRAF Mutation Analysis by Real Time PCR

CPT Codes
88381-Microdissection, manual.
81210-BRAF Mutation Analysis.

Indications for Ordering
To identify activating point mutations in the kinase domain of BRAF, an oncogene that is frequently mutated in many human malignancies including melanoma, colorectal cancer (CRC), papillary thyroid cancer, and lung cancer. BRAF mutations are found in sporadic microsatellite instability-high (MSI-H) CRC but not in CRC arising from HNPCC and therefore in concert with the hMLH1 methylation assay may be useful in determining germline versus sporadic MSI-H CRC. In addition, point mutations in BRAF may identify patients likely to benefit from treatment by BRAF inhibitors.

Methodology
The BRAF Mutation Assay detects the V600E/E2/D and V600K/R/M mutations in codon 600 of the BRAF gene using RealTime PCR. The BRAF Mutation Assay consists of three allele-specific duplex PCR reactions, designed to specifically amplify either the BRAF Wild Type, BRAF T1799>A and BRAF G1798>A mutations, each combined with an endogenous control gene that serves as a sample processing control. This control checks for adequate execution of the complete process from sample to result.

Specimen Types

  1. FFPE Tissue Blocks.
  2. FFPE Curls.
  3. Tissue Slides: 11 unstained
    (Collection Instructions: Submit 10 unstained, non-baked slides with 10-micron thick sections of the tumor tissue.)

Specimen Stability
Ambient (preferred)

Results

BRAF Mutation Assay can report 2 possible results:

  • No mutation detected in BRAF codon 600
  • Mutation detected in BRAF codon 600

Test Limitations
The BRAF Mutation Assay is designed to detect V600E, V600E2, V600D,V600K, V600R and V600M mutations in the BRAF gene. Due to its specific primer and probe design, V600E, V600E2 and V600D mutations cannot be distinguished from each other. This also applies for the V600K, V600R and V600M mutations. Other rare mutations, such as V600G, are not detected by the BRAF Mutation Assay.

The presence of PCR inhibitors, such as melanin, may cause a false negative result or an Insufficient DNA Input-result. The BRAF Mutation Assay is a qualitative test and is not intended for quantitative measurements of mutations. The supported tumor area for the BRAF Mutation Assay is 50 mm² for 5 μm tissue sections and 25 mm² for 10 μm tissue sections with a minimum tumor cell percentage of 50%.

Turnaround Time
24 hours

Test Disclaimer
This test was developed, and its performance characteristics determined by Medical College of Wisconsin in a manner consistent with CLIA/CAP requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA).

Microsatellite Instability Analysis Tumor PCR

Test Name
Microsatellite Instability Analysis (Tumor), PCR

CPT Codes
88381-Microdissection, manual.
81301-Microsatellite instability analysis.

Indications for Ordering
For determining microsatellite instability analysis (MSI) in colorectal and endometrial carcinomas for outcome predication and assessment of possibility hereditary contributions due to germline mutations in mismatch repair (MMR) genes. Tumors developing due to mutations in these genes exhibit instability of nucleotide repeats in microsatellites, which are short tandem repeat sequences distributed throughout the genome.

Methodology
The MSI assay is a fluorescence-based multiplex PCR assay for detection of microsatellite instability. Typically, MSI analysis involves comparison of allelic profiles of microsatellite markers generated by amplification of DNA from matching normal and tumor samples. Alleles present in the tumor sample that are not found in the corresponding normal samples indicate MSI. The current assay includes fluorescently-labeled primers for co-amplification of seven markers including five mononucleotides repeat markers6.1 (BAT-25, BAT-26, NR-21, NR-24 and NR-27) and two pentanucleotide repeat markers (Penta C and Penta D) (Table 1).

Table 1: The MSA Assay Loci information

[INSERT TABLE]

The amplified fragments are separated by capillary electrophoresis along with the internal size standards on the Applied Biosystems 3500 Genetic Analyzer. The data is analyzed with the GeneMapper Software. The mononucleotide markers are used for MSI determination and the pentanucleotide markers are used to detect potential sample mix ups and/or contamination. The mononucleotide repeat-markers included in the MSI assay were selected for high sensitivity and specificity to alterations in tumor samples with mismatch repair defects1. These mononucleotide-repeat markers are quasimonomorphic; that is, almost all individuals are homozygous for the same common allele for a given marker. Use of monomorphic markers simplifies data interpretation. The pentanucleotide repeat markers have been selected for their high level of polymorphism and low degree of MSI to uniquely identify samples, helping confirm that tumor and matching normal samples are from the same individual.

Specimen Required
Tumor and normal tissue are required. If normal tissue in a formalin-fixed, paraffin-embedded tissue block is not available, whole blood may be submitted.

Specimen Type

  1. FFPE Tissue Blocks.
  2. Tissue Slides (5-micrometer thick sections): 11 unstained of the tumor/normal tissue.
    (Collection Instructions: Submit 11 unstained, non-baked slides with 5-micron thick sections of the tumor tissue).
  3. Whole blood for normal: Container/Tube: lavender-top (EDTA)
    Specimen Volume: 3 mL
    Collection Instructions: Invert several times to mix blood.
    Send specimen in original tube.

Specimen Stability
Ambient (preferred)

Results

MSI Assay Results:

  • MSI-H (High)
  • MSI (Low)
  • MSS (Stable)

Test Limitations
Normal sample is needed to perform MSI assay.

Turnaround Time
7-8 business days

Test Disclaimer
This test was developed, and its performance characteristics determined by Medical College of Wisconsin in a manner consistent with CLIA/CAP requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA).

Solid Tumor-Targeted Cancer Gene panel by Next-Generation Sequencing, NGS

Test Name
Solid Tumor Targeted Cancer Gene Panel by Next-Generation Sequencing, NGS

CPT Codes
88381-Microdissection, manual.
81445-Targeted genomic sequence analysis panel, solid organ neoplasm.

Indications for Ordering

  • Identifying solid tumors that may respond to targeted therapies by assessing multiple gene targets simultaneously.
  • Identifying specific mutations within genes known to be associated with response or resistance to specific cancer therapies.
  • Identifying mutations that may help determine prognosis for patients with solid tumors.

Methodology
This test uses targeted next-generation sequencing (NGS) to evaluate for somatic mutations by amplifying 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes:

[INSERT TABLE]

This test is performed to evaluate for somatic mutations within solid tumor samples. This test is not intended for use for hematological malignancies. Additionally, this test does not assess for germline alterations within the genes listed.

Specimen Required
At least 10% tumor is required for this assay.

Specimen Type

Preferred: FFPE Tissue Blocks.

  1. Tissue Slides: 10 unstained non-baked slides with 10-micron thick sections of the tumor tissue.

Specimen Stability
Ambient (preferred)

Results
An interpretive report will be provided.

Test Limitations

  • Tumor samples adequacy.
  • This test cannot differentiate between somatic and germline alterations. Additional testing may be necessary to clarify the significance of results if there is a potential hereditary risk.
  • This test is not intended for use for hematological malignancies.
  • DNA variants of uncertain significance may be identified.
  • A negative (wild-type) result does not rule out the presence of a mutation that may be present but below the limits of detection of this assay (approximately 5%-10%).
  • This test does not detect large single or multi-exon deletions or duplications or genomic copy number variants.

Turnaround Time
12-15 business days

Test Disclaimer
This test was developed, and its performance characteristics determined by Medical College of Wisconsin in a manner consistent with CLIA/CAP requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA).

FLT3-ITD Mutation Analysis

Test Name
FLT3-ITD Mutation Analysis

CPT Codes
81245 FLT3 ITD mutation detection.

Indications for Ordering
A prognostic indicator in some acute myeloid leukemia patients.

Methodology
Genomic DNA is extracted from peripheral blood or bone marrow samples. A multiplex PCR is performed using fluorescent primers. The amplified product undergoes fragment analysis using capillary electrophoresis on the Applied Biosystems 3500 gene analyzer.

Specimen Required
At least 10% tumor is required for this assay.

Specimen Type

1. Peripheral blood (PB) or Bone Marrow (BM)

  • Container/Tube: EDTA (lavender top)
  • Specimen Volume: 3-5 mL

Collections Instructions:

  1. Invert several times to mix blood or BM.
  2. Send specimen in original tube.
  3. Label specimen with sample type (PB, BM).
  4. Specimen Stability: Ambient (preferred)/Refrigerate

2. Extracted DNA from blood or bone marrow

  • Container/Tube: 1.5- to 2-mL tube
  • Specimen Volume: Entire specimen

Collection Instructions: Label specimen as extracted DNA from blood or bone marrow and provide the volume and the DNA concentration as ng/ul.

Specimen Stability 
Frozen (preferred)/Refrigerate/Ambient

Results
An interpretive report will be provided indicating whether the FLT3 internal tandem duplication is detected or not.

Test Limitations
This test is not designed for monitoring residual disease following treatment.

Turnaround Time
3-5 business days

Test Disclaimer
This test was developed, and its performance characteristics determined by Medical College of Wisconsin in a manner consistent with CLIA/CAP requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration (FDA).

Data Analysis

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Whole Exome Sequencing Data Analysis

Test Name
Whole Exome Sequencing Data Analysis

CPT Code
81417

Test Information/Description
Prior arrangements must be made with the laboratory before submission of test request. Whole Exome Sequencing Data Analysis (WESDAT) is used to annotate variants in a patient's genome in order to determine the role of the genomic variants in disease outcomes and to determine the regions of the genome where there is no sequence coverage.

Methodology
Sequence variants are loaded into a tertiary analysis software package that contains data sources and algorithms allowing for the evaluation of whole exome sequencing variants for evolutionary conservation, predicted impact on protein structure and function, ability to disrupt conserved splice sites, and presence in databases including OMIM, dbSNP, and HGMD. The software package annotates variants with this data, taking into account both the reference gene model and any identified alternate transcripts. A custom software package is used to determine what parts of the exome are not sequenced. If Sanger confirmation of variants is requested, the DNA sequence of a segment of the genome surrounding each variant of interest is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy sequencing.

Test Limitations
WESDAT is validated to evaluate Single Nucleotide Variants (SNV) within the human genome. The ability to detect abnormal variants is dependent of the presence of SNVs in the data provided to the laboratory. Certain types of sequence variation are difficult to identify and have not been validated to be reliably detected for current clinical use. These include insertions, deletions, copy number variations, triplet repeat expansions, and structural chromosomal rearrangements.

Specimen Type
Data must be sent on an encrypted media source with the encryption method and password (send separately) Only one patient's data per media source is permitted. We cannot accept electronic data files for analysis. Data source will be returned upon request. WES data must be generated in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.

If Sanger sequencing of variants of interest is requested

  1. Whole Blood – 2-4mL of whole blood in a purple top EDTA (2 tubes per patient is preferred). For infants, we require a minimum of 1mL of blood. The tube(s) must be labeled with at least two patient identifiers. Ship blood tubes overnight at room temperature in an insulated container within 5 days of collection.
  2. gDNA – 10µg of purified gDNA with a minimum of 75ng/µL and a 260/280 purity ratio of 1.75-2.0 in a screw cap tube. The tube must be labeled with at least two patient identifiers. Ship gDNA overnight at room temperature. We do not accept products of genome amplification or other amplification reactions. DNA must be extracted in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.

Shipping Conditions
See above

Turn Around Time
30 Days

Whole Genome Sequencing Analysis

Test Name
Whole Genome Sequencing Data Analysis

CPT Code
81427

Test Information/Description
Prior arrangements must be made with the laboratory before submission of test request. Whole Genome Sequencing Data Analysis (WESDAT) is used to annotate variants in a patient's genome in order to determine the role of the genomic variants in disease outcomes and to determine the regions of the genome where there is no sequence coverage.

Methodology
Sequence variants are loaded into a tertiary analysis software package that contains data sources and algorithms allowing for the evaluation of whole genome sequencing variants for evolutionary conservation, predicted impact on protein structure and function, ability to disrupt conserved splice sites, and presence in databases including OMIM, dbSNP, and HGMD. The software package annotates variants with this data, taking into account both the reference gene model and any identified alternate transcripts. A custom software package is used to determine what parts of the exome are not sequenced. If Sanger confirmation of variants is requested, the DNA sequence of a segment of the genome surrounding each variant of interest is PCR amplified from purified genomic DNA followed by sequencing in the forward and reverse directions using automated fluorescent dideoxy sequencing.

Test Limitations
WGSDAT is validated to evaluate Single Nucleotide Variants (SNV) within the human genome. The ability to detect abnormal variants is dependent of the presence of SNVs in the data provided to the laboratory. Certain types of sequence variation are difficult to identify and have not been validated to be reliably detected for current clinical use. These include insertions, deletions, copy number variations, triplet repeat expansions, and structural chromosomal rearrangements.

Specimen Type
Data must be sent on an encrypted media source with the encryption method and password (send separately). Only one patient's data per media source is permitted. We cannot accept electronic data files for analysis. Data source will be returned upon request. WGS data must be generated in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.

If Sanger sequencing of variants of interest is requested

  1. Whole Blood – 2-4mL of whole blood in a purple top EDTA (2 tubes per patient is preferred). For infants, we require a minimum of 1mL of blood. The tube(s) must be labeled with at least two patient identifiers. Ship blood tubes overnight at room temperature in an insulated container within 5 days of collection.
  2. gDNA – 10µg of purified gDNA with a minimum of 75ng/µL and a 260/280 purity ratio of 1.75-2.0 in a screw cap tube. The tube must be labeled with at least two patient identifiers. Ship gDNA overnight at room temperature. We do not accept products of genome amplification or other amplification reactions. DNA must be extracted in a CLIA laboratory or a laboratory meeting equivalent requirements as determined by the CAP and /or CMS.

Shipping Conditions
See above

Turnaround Time
30 days

Cancellations

Test cancellation is permitted within 24 hours of receipt of the specimen by the Clinical Diagnostic Laboratory. Cancellation requests received after this time will not be honored. Test cancellations must be in writing by the ordering institution/provider via fax at (414) 955-6516. Tests will be performed, reported, and billed/invoiced if a test is not cancelled with proper documentation within 24 hours.

Privacy Statement

The Clinical Diagnostic Laboratory will disclose test results only to the ordering physician. Sequence data is stored on secured servers at the Medical College of Wisconsin. After six months, data is transferred to tape. The tapes are kept in secure storage. We will release data files (FASTQ, BAM, VCF) to patients and physicians with a signed consent form. This will incur a $50 fee. Data is distributed via an encrypted media device.