Important
To prevent destruction of tumor RNA by RNases from the hands, always wear gloves when handling tumor tissue. Undamaged tumor RNA is essential for a successful analysis.
The tumor biopsy must be taken WITHIN ONE HOUR of surgery.
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Taking the biopsy
To biopsy a standardized amount of tumor tissue, use the biopsy punch (diameter 3 mm) provided in the MammaPrint Specimen Collection and Transportation Kit. If this proves difficult, provide a good quality sample with a minimum diameter of 3 mm.
This video demonstrates how to sample and preserve a tumor biopsy to ensure that the subsequent MammaPrint analysis is successful. The need to follow the recommended sample preparation procedure is explained in relation to the analytical principles involved.
To start the video, please log in your Agendia web account. If you are not a registered user of the Agendia website, register here.
Surgical Specimen
D. de Jong, M.D. Ph.D
(Pathologist NKI - AVL Amsterdam)
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Core Biopsy
C.E. Loo, M.D.
(Radiologist NKI - AVL Amsterdam)
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Ensuring a good quality sample
When taking the biopsy, ensure that you have a good overview of the different tumor sections in order to obtain a representative sample. Sample should be taken from a region clear of necrotic and stromal tissue - the presence of these tissues reduces the number of tumor cells in the sample and may result in insufficient representative RNA.
Note that the centre of a tumor is often necrotic, while the edges of the tumor often contain many stromal cells. The best area to perform the biopsy is at the periphery of the tumor.
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Before you prepare your sample
Wear gloves!
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|
Remove the large tube from the kit
and open it.
Place the large screw cap and small tube on the table. Open the small tissue-sample tube. |
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Remove the biopsy punch from
package. |
Preparing your sample
A representative biopsy must be placed in the RNA-stabilizing solution within 1 hour of surgical excision.
|
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Use the biopsy punch provided to
obtain tumor sample with a minimum
thickness of 3 mm and a maximum
thickness of 5 mm.
Please ensure that the sample supplied
is taken from a region clear of necrotic
(middle of the tumor) and stromal tissue
(boarders of the tumor). |
Linking the sample ID to your patient
|
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Place the tissue sample in
preservative solution, and
tightly close the sample tube. |
|
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Peel off a bar code label
from the right corner of the
Test Request Form and place
it on the sample tube. |
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Place the small sample tube
into the large tube. |
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Peel off the second bar code
label from the Test Request
Form and place it on your
patient record. |
Completing the
Test Request Form (TRF)
Complete the information on the TRF and sign the
form:
- Record specimen collection date.
- Complete the healthcare provider’s information.
- Complete patient information, including name
and date of birth.
- Ensure appropriate clinical information is
provided, including the diagnosis code
(ICD-9 code).
- Ensure the ordering physician and/or submitting
pathologist has signed and dated the form.
- Ensure payment option is selected in the billing
payment section.
- If the insurance billing option is being selected,
ensure all patient contact information is
complete, including telephone number and
either of the following:
1. readable photocopies of front and back of
insurance card(s)
2. fill out insurance information completely.
- If patient payment option (self pay) is selected,
information must include either credit card
information or check/money order.
Packaging
|
|
Place the large tube into the
plastic bag.
Place the plastic bag into
the Specimen Collection and
Transportation Kit (box). |
Place the following items into
the box:
- Completed TRF - first page
should have been faxed to
Agendia at (888)-669-3292.
- Photocopies of patient’s
insurance card(s) if applicable.
- Check or money order if
patient paid out of pocket.
Shipping
Ship at ambient temperature, do not freeze sample or use dry ice for shipment.
|
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Put the Specimen Collection
and Transportation Kit into
the FedEx Clinical Pak.
On the FedEx prepared airbill:
Complete section 1 (“From”)
Sign the section 9
(“Required Signature”)
Insert the completed FedEx airbill and
3 copies of the Commercial Invoice
(printed on paper from your institute)
in the document bag. Keep the FedEx
Customer receipt and original copy of the
Commercial Invoice.
|
Schedule the Package Pick-up
Call FedEx at (800)-247-4747 to schedule
a package pick-up.
Questions
For more information, go to www.agendia.com.
You can contact Agendia via e-mail:
This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
or call 888-321-2732.
Downloads
MammaPrint - Sampling and Shipping Instructions0.22 MB
Gene expression profiling
MammaPrint analyses the expression of 70 pre-selected genes in breast tumor samples, comparing their activity with reference samples of ‘Low Risk’ and ‘High Risk’ profiles. The principles of gene expression profiling and the DNA microarray technology used to determine an expression profile are described here.
Confidence and quality
Agendia uses industry-quality, customized microarrays, manufactured by Agilent Technologies - one of the world's leading suppliers of microarray products. Each microarray contains three identical sets of the 70 genes to be analyzed. This enables three independent measurements of the 70-gene expression profile, increasing confidence in the test result.
In addition, each array contains several hundred, carefully selected normalization and control genes as well as negative control genes (DNA sequences to which no human mRNA can bind) to enable the monitoring of various technical aspects of the microarray process.
In total, more than 25 Quality Control parameters are implemented within the test process to ensure the correct result.
MammaPrint analysis – in brief
Preliminary examination
Each tumor biopsy is reviewed for histology and RNA quality to ensure that the sample is in a suitable condition for MammaPrint analysis. If the sample fails to meet predetermined criteria, for example comprising < 30% tumor cells, it will be rejected and the physician or pathologist will be notified immediately. In this case, there will be no fee.
Preparation
The tissue biopsy is frozen to cut tissue sections for pathology
examination and RNA isolation. RNA is extracted, amplified and labeled
with Cy3 and Cy5 dyes using standard protocols, RNA from a reference is
similarly amplified and labeled.
Hybridization
The sample is labeled with both Cy3 and Cy5 and hybridized against a Cy5 or Cy3 labeled reference. The duplicate is hybridized onto the microarray with the 70 genes.
Scanning and evaluation
The fluorescent intensity of the individual spots on the microarray is a measure of the activity of a particular gene. Each spot is measured twice, one measurement for each dye (Cy3 and Cy5).
Feature Extraction Software determines the intensities of the individual features for both channels, subtracts background signal, performs normalization, and calculates ratios, errors and p values for each spot.
X-Print Analysis® software further analyzes the data according to a specific algorithm that determines the MammaPrint Index and thereby the expression profile of the sample (Low Risk, High Risk). An internal report is produced for each sample, specifying quality control values and results.
Classification
Tumor samples with a MammaPrint Index above the threshold of 0.4 are classified as Low Risk, and tumor samples with a MammaPrint Index equal to or smaller than the threshold are classified as High Risk.
In the very rare case of a borderline result index (between and including 0.365 and 0.435), the sample will be re-labeled, re-hybridized, and re-analyzed. The average MammaPrint Index of both experiments will be calculated. The sample will be reported as borderline if the index is between the borderline region for two measurements (between and including 0.3775 and 0.4225).
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