AlloMap Heart Molecular Expression Testing
Device Facts
| Record ID | K221640 |
|---|---|
| Device Name | AlloMap Heart Molecular Expression Testing |
| Applicant | Caredx, Inc. |
| Product Code | OJQ · Cardiovascular |
| Decision Date | Sep 13, 2023 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 862.1163 |
| Device Class | Class 2 |
Indications for Use
AlloMap Heart Molecular Expression Testing is an In Vitro Diagnostic Multivariate Index assay (IVDMIA) test service, performed in a single laboratory, assessing the gene expression profile of RNA isolated from peripheral blood mononuclear cells (PBMC). AlloMap Heart Testing is intended to aid in the identification of heart transplant recipients with stable allograft function who have a low probability of moderate/severe acute cellular rejection (ACR) at the time of testing in conjunction with standard clinical assessment.
Device Story
IVDMIA test service assessing gene expression profile of RNA from peripheral blood mononuclear cells (PBMC). Physician orders blood draw into CPT tubes; PBMCs separated, washed, pelleted, and lysed at referring facility; lysate shipped to CareDx laboratory. Laboratory purifies RNA; performs qRT-PCR on 20 genes (11 informative, 9 for normalization/QC) using Roche LightCycler 480 II instruments. CareDx Analyzer software converts expression levels into AlloMap Test Score (0-40). Results reported to physician for clinical interpretation alongside standard assessments to determine probability of acute cellular rejection (ACR).
Clinical Evidence
Bench testing only. Method comparison study using 163 patient RNA samples compared candidate device (Roche LC480) to predicate (ABI 7900HT). Passing-Bablok regression showed slope of 1 (95% CI 0.99-1.01) and intercept of 0. ATD analysis showed >98% of samples within acceptable zones. NPV/PPV differences between candidate and predicate were <1% and <2% respectively. Precision studies (N=90-144 replicates per pool) demonstrated acceptable within-lab and between-instrument variability.
Technological Characteristics
In Vitro Diagnostic Multivariate Index Assay (IVDMIA). Analyzes 20-gene expression via qRT-PCR. Instrumentation: Roche LightCycler 480 II. Software: CareDx Analyzer. Sample input: PBMC-derived RNA (min 400 ng). Output: Integer score (0-40). Calibration: None required. QC: Automated checks on every sample. Laboratory-based service (CLIA-certified/CAP-accredited).
Indications for Use
Indicated for heart transplant recipients 15 years of age or older, at least 2 months (≥55 days) post-transplantation.
Regulatory Classification
Identification
A cardiac allograft gene expression profiling test system is a device that measures the ribonucleic acid (RNA) expression level of multiple genes and combines this information to yield a signature (pattern, classifier, index, score) to aid in the identification of a low probability of acute cellular rejection (ACR) in heart transplant recipients with stable allograft function.
Special Controls
*Classification.* Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Cardiac Allograft Gene Expression Profiling Test Systems.” See § 862.1(d) for the availability of this guidance document.
Predicate Devices
Related Devices
- DEN080007 — ALLOMAP MOLECULAR EXPRESSION TESTING · Xdx · Aug 26, 2008
- K181661 — QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System · Bio-Rad Laboratories, Inc. · Feb 13, 2019
Submission Summary (Full Text)
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FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY AND INSTRUMENT
## I Background Information:
A 510(k) Number
K221640
B Applicant
CareDx, Inc.
C Proprietary and Established Names
AlloMap Heart Molecular Expression Testing
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| OJQ | Class II | 21 CFR 862.1163 - Cardiac Allograft Gene Expression Profiling Test System | CH - Clinical Chemistry |
## II Submission/Device Overview:
A Purpose for Submission:
Modification of the AlloMap® Molecular Expression Testing (DEN080007/k073482) to change the RT-PCR instruments used as components of the device
B Measurand:
RNA expression of 20 genes
C Type of Test:
Quantitative RT-PCR analysis of 20 genes performed in a single laboratory at CareDx, Inc. in Brisbane, CA
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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K221640 - Page 2 of 9
## III Intended Use/Indications for Use:
### A Intended Use(s):
See Indications for Use below.
### B Indication(s) for Use:
AlloMap Heart Molecular Expression Testing is an In Vitro Diagnostic Multivariate Index assay (IVDMIA) test service, performed in a single laboratory, assessing the gene expression profile of RNA isolated from peripheral blood mononuclear cells (PBMC). AlloMap Heart Testing is intended to aid in the identification of heart transplant recipients with stable allograft function who have a low probability of moderate/severe acute cellular rejection (ACR) at the time of testing in conjunction with standard clinical assessment.
Indicated for use in heart transplant recipients:
- 15 years of age or older
- At least 2 months (≥55 days) post-transplantation
### C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
### D Special Instrument Requirements:
Roche LightCycler 480 II (qualified by CareDx, Inc.), which replace the RT-PCR instruments used with the device in DEN080007/k073482.
## IV Device/System Characteristics:
### A Device Description:
AlloMap Heart Molecular Expression Testing is an In Vitro Diagnostic Multivariate Index Assay (IVDMIA) test service performed in a single laboratory. It assesses the gene expression profile of RNA isolated from peripheral blood mononuclear cells (PBMC) using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Gene expression levels are converted to an AlloMap Test Score, which is reported to the physician.
The following sample collection supplies are provided by CareDx to ordering laboratories/physicians:
- 8 mL BD Vacutainer® CPT™ Cell Preparation Tubes with Sodium Citrate (CPT) (only tubes supplied by CareDx should be used)
- AlloMap sample processing reagent kit (10 centrifuge tubes containing 5 mL phosphate buffered saline, 10 tubes containing 1.8 mL of LyseDx™ lysing reagent, 10 sterile disposable transfer pipettes, 1 package insert with instructions for use, 1 quick reference card)
- CareDx Frozen Shipper Pack
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# Principle of Operation:
Blood specimen collection at the referring facility is initiated by a physician order. Whole blood is drawn from the patient into blood collection tubes supplied by CareDx. PBMCs are separated from whole blood via centrifugation, washed, pelleted, and lysed using reagents supplied by CareDx. The lysate is stored frozen and shipped to the CareDx laboratory using kits provided by CareDx. Upon arrival at the CareDx laboratory, samples are inspected and accessioned into the Laboratory Information Management System (LIMS) database that tracks all workflow steps.
RNA from the PBMC lysate is purified and quantified with a spectrophotometer. Upon meeting RNA quantity and purity acceptance criteria, liquid handling instruments are used to dispense samples and reagents into 96-well reaction plates. RNA is converted to cDNA via a reverse transcriptase protocol on a thermal cycler. The cDNA is then diluted and dispensed using liquid handling instruments into 384-well AlloMap plates containing qRT-PCR reagents. qRT-PCR of 20 genes (11 informative and 9 used for normalization and quality control) is carried out using qRT-PCR instruments. Multiple automated quality control (QC) checks are performed on every sample.
For all samples that pass QC, CareDx Analyzer software converts the expression levels determined for the 20 genes into an AlloMap Test Score (an integer ranging from 0 to 40) using a proprietary algorithm. Final reports are signed off by the clinical laboratory scientists in the CareDx laboratory and are sent to the ordering physician. The physician interprets the results along with other standard clinical assessments of cardiac allograft status to determine probability of ACR.
AlloMap Heart Molecular Expression Testing is only performed at the CLIA-certified and CAP-accredited clinical laboratory at CareDx in Brisbane, CA.
# B Instrument Description Information:
1. Instrument Name:
Roche LightCycler 480 (LC480) RT-PCR instruments are replacing the Applied Biosystems 7900HT RT-PCR instruments. The CareDx Analyzer software has been modified to support the LC480 instruments.
2. Specimen Identification:
The specimen identification method remains the same as the predicate device (DEN080007/k073482).
3. Specimen Sampling and Handling:
The specimen sampling and handling procedures remain the same as the predicate device (DEN080007/k073482).
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4. Calibration:
The calibration (i.e., no user calibration is required) remains the same as the predicate device (DEN080007/k073482).
5. Quality Control:
The quality control procedures and criteria remain the same as the predicate device (DEN080007/k073482).
V Substantial Equivalence Information:
A Predicate Device Name(s):
Allomap Molecular Expression Testing
B Predicate 510(k) Number(s):
DEN080007/K073482
C Comparison with Predicate(s):
| Device & Predicate Device(s): | K221640 | K073482 |
| --- | --- | --- |
| Device Trade Name | AlloMap Heart Molecular Expression Testing | AlloMap® Molecular Expression Testing |
| General Device Characteristic Similarities | | |
| Intended Use | Intended for assessing the gene expression profile of RNA isolated from PBMC to aid in the identification of heart transplant recipients with stable allograft function who have a low probability of moderate/severe accurate cellular rejection (ACR) at the time of testing in conjunction with standard clinical assessment. | Same |
| Indicated population | Heart transplant recipients 15 years of age or older and at least 2 months (≥ 55 days) | Same |
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| | post-transplant | |
| --- | --- | --- |
| Methodology | qRT-PCR performed in the CareDx laboratory to analyze a panel of 20 genes used to calculate an AlloMap test score | Same |
| General Device Characteristic Differences | | |
| qRT-PCR instruments | Roche LightCycler 480II (LC480) | Applied Biosystems (ABI) 7900HT |
| Analysis Software | Converts sample data generated by LC480 instruments to an AlloMap test score | Converts sample data generated by ABI 7900HT instruments to an AlloMap test score |
VI Standards/Guidance Documents Referenced:
CLSI EP09c 3rd Edition: Measurement Procedure Comparison and Bias Estimation Using Patient Samples.
CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition.
FDA guidance document “Cardiac Allograft Gene Expression Profiling Test Systems – Class II Special Controls Guidance for Industry and FDA Staff” issued on October 21, 2009.
FDA guidance document “Assay Migration Studies for In Vitro Diagnostic Devices” issued on April 25, 2013.
VII Performance Characteristics (if/when applicable):
A Analytical Performance:
1. Precision/Reproducibility:
A study was conducted to determine the precision of the AlloMap Heart Molecular Expression Testing. Three cDNA pools created from RNA isolated from blood samples from heart transplant patients and that cover the range of AlloMap scores (i.e., 16.2 (low), 28.5 (medium), and 37.5 (high)) were tested using two operators over five non-consecutive days. On each day, each cDNA pool was tested in each of six sections per plate and on each of three plate lots. The three plates for each pool were tested across three LC480 instruments. A total of 90 replicates (6 replicates/plate x 3 plates/day x 5 days) was conducted per pool and replicates that passed established quality control metrics were included in the analysis. The results of the testing are shown below.
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| | Low pool N = 88 | | Medium pool N = 90 | | High pool N = 90 | |
| --- | --- | --- | --- | --- | --- | --- |
| AlloMap score | 16.2 | | 28.5 | | 37.5 | |
| Component | SD | %CV | SD | %CV | SD | %CV |
| Between day | 0.762 | 4.7 | 0.222 | 0.8 | 0.000 | 0.0 |
| Between operators | 0.835 | 5.1 | 0.166 | 0.6 | 0.000 | 0.0 |
| Between instruments | 0.000 | 0.0 | 0.000 | 0.0 | 0.024 | 0.1 |
| Between plate lots | 0.518 | 3.2 | 0.000 | 0.0 | 0.148 | 0.4 |
| Between plates within a lot | 0.690 | 4.3 | 0.289 | 1.0 | 0.087 | 0.2 |
| Between plate sections | 0.636 | 3.9 | 0.319 | 1.1 | 0.166 | 0.4 |
| Repeatability | 1.567 | 9.7 | 0.984 | 3.4 | 0.259 | 0.7 |
| Within-lab | 2.210 | 13.6 | 1.110 | 3.9 | 0.353 | 0.9 |
An additional study was conducted to establish between-instrument variability among the eight LC480 instruments qualified by the CareDx laboratory for the candidate device. Three cDNA pools created from RNA isolated from blood samples from heart transplant patients and that cover the range of AlloMap scores (i.e., 17.5 (low), 29.0 (medium), and 34.5 (high)) were tested. Each cDNA pool was tested in each of six sections per plate and on each of three plates from one lot of plates. The three plates for each pool were tested on each of eight LC480 instruments for a total of 144 replicates (6 sections/plate x 3 plates/instrument x 8 instruments) per pool. Replicates that passed established quality control metrics were included in the analysis. Between-instrument results along with between-plate variability, repeatability, and total variability results are shown below.
| | Low pool N = 142 | | Medium pool N = 144 | | High pool N = 144 | |
| --- | --- | --- | --- | --- | --- | --- |
| AlloMap score | 17.5 | | 29.0 | | 34.5 | |
| Component | SD | %CV | SD | %CV | SD | %CV |
| Between instruments | 0.31 | 1.77 | 0.18 | 0.61 | 0.10 | 0.30 |
| Between plates | 0.19 | 1.08 | 0.05 | 0.16 | 0.12 | 0.35 |
| Repeatability | 1.35 | 7.71 | 0.96 | 3.31 | 0.57 | 1.64 |
| Total | 1.40 | 7.98 | 0.98 | 3.37 | 0.59 | 1.70 |
2. Linearity:
Linearity is not applicable. AlloMap Heart Molecular Expression Testing provides physicians with a score between 0 and 40.
3. Analytical Specificity/Interference:
The analytical specificity for AlloMap Heart Molecular Expression Testing was established in DEN080007/k073482.
4. Assay Reportable Range:
AlloMap Heart Molecular Expression Testing provides physicians with a score between 0 and 40.
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5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
Quality control materials and procedures remain unchanged from DEN080007/k073482.
Sample stability study protocols and data were reviewed and found acceptable. Patient RNA samples are stored for up to 6 months at ≤ -65°C and patient cDNA samples are stored for up to 6 months at ≤ -15°C. RNA controls are stored for up to 16 months at ≤ -65°C and cDNA controls are stored for up to 45 months at -15°C to -35°C.
6. Detection Limit:
A study was conducted to confirm that a minimum RNA yield of 400 ng is required to run the assay. A pool of patient RNA samples with an AlloMap score of 34.6 was tested in RNA inputs ranging from 150 to 500 ng to generate cDNA. Each cDNA was tested three times in the same plate section across three plates from the same lot using one LC480 instrument. The results confirmed the minimum RNA yield of 400 ng.
As established in DEN080007/k073482, the blood collection procedure yields sufficient RNA to run the AlloMap test on 98% of patient samples, the acceptable A₂₆₀/A₂₈₀ ratio range for testing is 1.5 to 3.5, and each gene assay must provide a C_T at or below 38 to generate a test score.
7. Assay Cut-Off:
Not applicable.
8. Accuracy (Instrument):
See the method comparison with predicate device study in section VII.B.1.
9. Carry-Over:
Not applicable.
B Comparison Studies:
1. Method Comparison with Predicate Device:
A method comparison study was conducted using 163 heart transplant patient RNA samples spanning the range of AlloMap scores. cDNA was generated from each RNA sample and each cDNA was tested on two candidate devices (with Roche LightCycler 480II (LC480) instruments) and one predicate device (with Applied Biosystems Incorporated 7900HT (7900HT) instruments).
Passing-Bablok regression analysis was conducted with the AlloMap scores from each pair of predicate and candidate devices used in the method comparison study. The results were as follows:
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| Comparison | Slope estimate (95% confidence interval) | Intercept estimate (95% confidence interval) | R² |
| --- | --- | --- | --- |
| Candidate device 1 vs. predicate device | 1 (0.99, 1.01) | 0 (-0.53, 0.53) | 0.9603 |
| Candidate device 2 vs. predicate device | 1 (0.99, 1.01) | 0 (-034, 0.34) | 0.9512 |
An allowable total difference (ATD) analysis was conducted with the method comparison data and within-laboratory imprecision estimates for the predicate device according to FDA’s guidance document “Assay Migration Studies for In Vitro Diagnostic Devices” (issued on April 25, 2013). The analysis showed that over 98% of the samples over the entire AlloMap score range fell within the ATD zones with the lower limit of the 95% one-sided confidence interval being above 94% for both candidate devices tested. Therefore, it was concluded that the candidate device performance is comparable to the predicate device performance since the majority of the candidate device measurements fall within the imprecision of the predicate device.
Analyses were provided that demonstrated that the mean NPV and PPV differences between the candidate and predicate devices were within 1% for NPV and within 2% for PPV for both the 2-6 month and > 6 month post-transplant periods.
2. Matrix Comparison:
Not applicable.
C Clinical Studies:
1. Clinical Sensitivity:
See section C.3. below.
2. Clinical Specificity:
See section C.3. below.
3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
Clinical performance of AlloMap Heart Molecular Expression Testing in cardiac transplant patients was established in DEN080007/k073482.
D Clinical Cut-Off:
See section C.3. above.
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E Expected Values/Reference Range:
AlloMap Heart Molecular Expression Testing provides physicians with a score between 0 and 40. This is unchanged from the predicate device (DEN080007/k073482).
F Other Supportive Instrument Performance Characteristics Data:
Not applicable.
VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
