(418 days)
B·R·A·H·M·S™ CgA II KRYPTOR™ is an automated immunofluorescent assay using Time-Resolved Amplified Cryptate Emission (TRACE™) technology for quantitative determination of Chromogranin A concentration in human serum.
B·R·A·H·M·S™ CgA II KRYPTOR™ is to be used in conjunction with other clinical methods as an aid in monitoring of disease progression during the course of disease and treatment in patients with gastroentero-pancreatic neuroendocrine tumors (GEP-NETs, grade 1 and grade 2).
The B-R-A-H-M-S CgA II KRYPTOR assay is based on the formation of a complex comprised of a Chromogranin A (CgA) analyte "sandwiched" between two monoclonal mouse anti-CgA antibodies. One of the antibodies (537/H2) is directed at the epitope AA124–144 and labelled with DiSMP cryptate, the other antibody (541/E2) binds to AA280-301 and is labelled with Alexa Fluor®647.
The measurement principle is based on a non-radiative energy transfer from a donor (cryptate) to an acceptor (Alexa Fluor™647) when they are part of an immunocomplex (TRACE technology (Time-Resolved Amplified Cryptate Emission)).
The fluorescent signal is proportional to the concentration of the analyte to be measured.
With this principle B-R-A-H-M-S CgA II KRYPTOR is a homogenous one-step immunoassay for the quantification of CgA II in human serum. The linear direct measuring range of the assay is from 20-3,000 ng/mL, going up to 1,000,000 ng/mL with automated dilution. Results can be retrieved after a 29 min incubation time.
Here's an analysis of the acceptance criteria and study findings for the B.R.A.H.M.S CgA II KRYPTOR device, based on the provided FDA 510(k) summary:
Acceptance Criteria and Reported Device Performance
Note: The provided document primarily describes analytical performance criteria and clinical performance measures (sensitivity, specificity) rather than explicit "acceptance criteria" in a pass/fail format for clinical decision-making. However, the sensitivity and specificity values obtained from the clinical study serve as the reported device performance against which implicit clinical acceptance would be judged. The analytical performance metrics are generally presented as numerical results meeting industry standards (CLSI guidelines).
| Acceptance Criteria Category | Specific Metric | Acceptance Threshold (Implicit/Standard) | Reported Device Performance |
|---|---|---|---|
| Analytical Performance | Precision (Repeatability CV) | Generally low CVs for quantitative assays (e.g., <10% for low concentrations, <5% for higher) | Low range (23.0 ng/mL): 5.2% High range (2687 ng/mL): 1.6% |
| Precision (Within-Laboratory CV) | Generally low CVs for quantitative assays (e.g., <10% for low concentrations, <5% for higher) | Low range (23.0 ng/mL): 10.0% High range (2687 ng/mL): 7.4% | |
| Precision (Lot-to-Lot CV) | Generally low CVs (e.g., <5%) | Low range (26.3 ng/mL): 1.2% High range (2895 ng/mL): 0.0% (Note: Some 0.0% values may indicate very low variability or rounding) | |
| Precision (Reproducibility CV) | Generally low CVs (e.g., <10% for low concentrations, <5% for higher) | Low range (25.7 ng/mL): 9.0% High range (92,561 ng/mL): 5.6% | |
| Limit of Blank (LoB) | As low as technically feasible, ensuring differentiation from zero. | 11.3 ng/mL | |
| Limit of Detection (LoD) | As low as technically feasible, ensuring reliable detection of low concentrations. | 14.0 ng/mL | |
| Limit of Quantitation (LoQ) | Lowest concentration with a within-laboratory precision CV of ≤ 20%. | 20.0 ng/mL (met ≤ 20% CV) | |
| Linearity Range | Demonstrated across the claimed measuring range. | 20.0 ng/mL (LoQ) up to 1,000,000 ng/mL (with dilution) | |
| Dilution Recovery | Typically recovery within 85-115% of expected values. | Mean recovery values ranging from 97.6% - 109.6% | |
| Spike Recovery | Typically recovery within 90-110% of expected values. | Individual recovery values ranging from 91% - 109% | |
| High Dose Hook Effect | Absent or managed by automatic detection/dilution. | Detected by kinetics analysis; automatic dilution for samples > 3,000 ng/mL, extending range up to 1,000,000 ng/mL. | |
| Interference | Bias ≤ 10% for common endogenous and exogenous interfering substances. | Substances evaluated were found not to affect test performance (bias ≤ 10%) at clinically relevant concentrations. | |
| Cross-Reactivity | Low cross-reactivity with structurally similar substances. | Between -21.6% - 0.03% (for various CgA fragments and related proteins). | |
| Clinical Performance | Clinical Sensitivity (for tumor progression based on ΔCgA > 50% & >100 ng/mL cutoff) | Sufficient to aid monitoring, balancing with specificity given the intended use (aid, not standalone diagnosis). | 34.4% (95% CI: 23.2% - 45.5%) |
| Clinical Specificity (for tumor progression based on ΔCgA > 50% & >100 ng/mL cutoff) | Sufficient to aid monitoring, balancing with sensitivity given the intended use (aid, not standalone diagnosis). | 93.4% (95% CI: 90.2% - 96.0%) | |
| Positive Predictive Value (PPV) | Relevant for clinical utility given prevalence. | 57.9% (95% CI: 40.5% - 73.6%) | |
| Negative Predictive Value (NPV) | Relevant for clinical utility given prevalence. | 84.3% (95% CI: 79.3% - 89.1%) |
Study Details:
-
Sample size used for the test set and the data provenance:
- Clinical Study (for Sensitivity and Specificity): 153 adult GEP-NET patients (grade 1 and 2), with 459 total observations (likely reflecting multiple monitoring visits per patient). The study was described as a prospective study.
- Clinical Cut-off Derivation: 102 patients with diagnosed well-differentiated G1 and G2 GEP-NETs. This was a retrospective, bicentric observational pilot study.
- Reference Range Determination: 206 samples from self-declared healthy individuals. Data provenance is USA.
- Analytical studies: Various sample sizes were used, often involving replicates of pooled or individual human serum samples. For example, LoQ used 420 total replicates from 7 different pools of human serum samples.
- Provenance for analytical samples: Not explicitly stated but generally implied to be from human subjects, for instance, "human serum samples".
-
Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience):
- For the clinical study, tumor progression was classified by RECIST 1.1 criteria. This implies that experts (typically radiologists or oncologists) were involved in interpreting imaging (CT/MRI) according to these established criteria to determine the ground truth for tumor progression.
- The document does not specify the direct number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience"). However, RECIST 1.1 is an internationally recognized standard for evaluating cancer treatment response based on imaging, implying adjudication by qualified personnel.
-
Adjudication method (e.g. 2+1, 3+1, none) for the test set:
- The ground truth for tumor progression in the clinical studies was established using RECIST 1.1 criteria based on standard imaging (CT/MRI).
- The document does not explicitly state an adjudication method like "2+1" or "3+1" for discordant interpretations if multiple readers were involved in RECIST assessment. However, RECIST guidelines themselves are designed to standardize interpretation, and clinical trials often employ independent central review or consensus panels for definitive RECIST ratings, though this specific detail is not provided here.
-
If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) for quantitative determination of Chromogranin A concentration in human serum, intended to be used in conjunction with other clinical methods as an aid in monitoring. It is not an AI-assisted imaging device or a device that directly aids human readers in interpreting images.
-
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- This is an IVD assay, which functions as a "standalone" measurement of a biomarker in serum. The results are generated by the automated instrument (B.R.A.H.M.S KRYPTOR compact PLUS analyzer) without direct human interpretation of the measurement itself. However, the device's output (CgA concentration) is explicitly stated to not be used for standalone diagnosis or monitoring but "in conjunction with other clinical methods." So while the analytical measurement is standalone, the clinical interpretation for decision-making is not.
-
The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
- For the clinical performance evaluation (sensitivity and specificity for tumor progression), the ground truth was imaging-based tumor assessment using RECIST 1.1 criteria. This is a form of expert assessment based on a standardized methodology, often relying on radiologists and oncologists to interpret imaging studies.
-
The sample size for the training set:
- This document describes an IVD device submission, not a machine learning/AI device. Therefore, the concept of a "training set" for an algorithm in the typical AI sense does not directly apply. The development and validation of the assay itself would have involved numerous samples for optimization and establishment of analytical performance characteristics, but these are not referred to as a "training set" here.
-
How the ground truth for the training set was established:
- As addressed above, the concept of a "training set" in the context of machine learning/AI is largely inapplicable here. The development of the assay's analytical characteristics (e.g., linearity, precision, detection limits) would be established through standard laboratory practices and reference materials, for which "ground truth" is defined by known concentrations or established analytical methods.
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September 18, 2023
B. R. A.H. M.S GmbH, part of Thermo Fisher Scientific Anne Kummerow Regulatory Affairs Specialist Neuendorfstrasse 25 Hennigsdorf, Brandenburg 16761 Germany
Re: K222251
Trade/Device Name: B.R.A.H.M.S CgA II KRYPTOR B.R.A.H.M.S CgA II KRYPTOR CAL B-R-A-H-M-S CgA II KRYPTOR QC Regulation Number: 21 CFR 866.6010 Regulation Name: Tumor-associated antigen immunological test system Regulatory Class: Class II Product Code: QXS Dated: May 11, 2023 Received: May 11, 2023
Dear Anne Kummerow:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
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Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.
For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely.
Ying Mao -S
Ying Mao, Ph.D. Branch Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Ouality Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K22251
Device Name B·R·A·H·M·S™ CgA II KRYPTOR™
Indications for Use (Describe)
B · R · A · H · M · S™ C g A II KRYPTOR™ is an automated immunothuorescent assay using Time-Resolved Amplified Cryptate Emission (TRACE™) technology for quantitative determination of Chromogranin A concentration in human serum.
B R A H M S M C g A II KRYPTOR™ is to be used in conjunction with other clinical methods as an aid in monitoring of disease progression during the course of disease and treatment in patients with gastroentero-pancreatic neuroendocrine tumors (GEP-NETs, grade 1 and grade 2).
X Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)
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510(k) Summary
807.92(a)(1), (2), (3)
The assigned 510(k) Number: K222251
1. Applicant
BRAHMS GmbH, part of Thermo Fisher Scientific Neuendorfstr. 25 16761 Hennigsdorf, Germany
| Contact: | Anne KummerowRegulatory Affairs Specialist |
|---|---|
| Phone: | +49 (0) 3302 883 2218 |
| Fax: | +49 (0) 3302 883 919 |
| E-mail: | anne.kummerow@thermofisher.com |
Date prepared: September 14th, 2023
2. Purpose for Submission
New Device
3. Device Trade Name
B·R·A·H·M·S™ CgA II KRYPTOR™
4. Regulatory Information
A Regulation section:
- 21 CFR 866.6010 Tumor-associated antigen immunological test system
- 21 CFR 862.1660 Quality Control material (assayed and unassayed)
- 21 CFR 862.1150 Calibrator
- 21 CFR 866.4520 Immunofluorometer equipment
- B Classification: Assay: Class II
- C Product code:
QXS, Chromogranin A (CgA)
[Class II]
- D Panel: Immunology (82) (Assay) Clinical Chemistry (75) (Calibrator and Controls)
5. Intended Use 807.92(a)(5)
- A. Intended use(s):
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B·R·A·M·S™ CgA II KRYPTOR™ is an automated immunofluorescent assay using Time-Resolved Amplified Cryptate Emission (TRACE™) technology for quantitative determination of Chromogranin A concentration in human serum.
B·R·A·M·S™ CgA II KRYPTOR™ is to be used in conjunction with other clinical methods as an aid in monitoring of disease progression during the course of disease and treatment in patients with gastroentero-pancreatic neuroendocrine tumors (GEP-NETs, grade 1 and grade 2).
B. Warnings
B-R-A-H-M-S CqA II KRYPTOR should not be used for cancer screening or cancer diagnosis.
B-R-A-H-M-S CgA II KRYPTOR is not indicated to be used as a stand-alone diagnostic monitoring assay and should be used in conjunction with clinical signs and symptoms and other diagnostic evidence. In cases where the laboratory results do not agree with the clinical picture or history, additional tests should be performed.
The results reported by the laboratory to the physician must include the identity of the Chromogranin A assay used. Values obtained with different assay methods cannot be used interchangeably. If, in the course of monitoring a patient, the assay method used for determining Chromogranin A levels is changed, additional tests should be carried out to determine the baseline values.
High levels of Chromogranin A (CgA) could also be found in cases of benign diseases (such as gastro-intestinal disorders, kidney failure and cardiovascular disorders) and in cancers other than NETs (such as adenocarcinoma of the breast, lung, or colon) [1-6]. CgA values may rise during treatment with proton pump inhibitors.
C. Limitations
The effect of interfering substances has only been evaluated for those listed in the labeling. Interference by substances other than those described in the Interference section below could lead to erroneous results.
Accurate results are dependent on following the proper sample collection, storage, and handling procedures.
- D. Special Conditions for use statement(s): Prescription use only
- E. Special Instruments requirements: B·R·A·H·M·S KRYPTOR compact PLUS analyzer
6. Device Description 807.92(a)(4)
The B-R-A-H-M-S CgA II KRYPTOR assay is based on the formation of a complex comprised of a Chromogranin A (CgA) analyte "sandwiched" between two monoclonal mouse anti-CgA antibodies. One of the antibodies (537/H2) is directed at the epitope AA124–144 and labelled with DiSMP cryptate, the other antibody (541/E2) binds to AA280-301 and is labelled with Alexa Fluor®647.
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The measurement principle is based on a non-radiative energy transfer from a donor (cryptate) to an acceptor (Alexa Fluor™647) when they are part of an immunocomplex (TRACE technology (Time-Resolved Amplified Cryptate Emission)).
The fluorescent signal is proportional to the concentration of the analyte to be measured.
With this principle B-R-A-H-M-S CgA II KRYPTOR is a homogenous one-step immunoassay for the quantification of CgA II in human serum. The linear direct measuring range of the assay is from 20-3,000 ng/mL, going up to 1,000,000 ng/mL with automated dilution. Results can be retrieved after a 29 min incubation time.
7. Substantial Equivalence Information
- a. Predicate Device Name(s)/510(k) number(s)/product code(s): ARCHITECT CEA / K990774 / DHX
- b. Reference Device Name(s) 510(k) number(s)/product code(s): B-R-A-H-M-S™ PCT sensitive KRYPTOR™ / B-R-A-H-M-S™ KRYPTOR™ compact PLUS / K171338 / PMT, PRI, NMT, JZT
6.1 Technological Characteristics 807.92(a)(6):
c. Comparison with predicate:
The intended use and fundamental scientific technology between the predicate device and the new device are substantially equivalent. Comparison of the B-R-A-H-M-S CgA II KRYPTOR to a well-established clinical comparator method based on tumor imaging and gold standard evaluation by RECIST 1.1 criteria met the predefined clinical performance criteria for sensitivity and specificity in detecting tumor progressions.
| Item | Subject Device: | Predicate Device:K990774 |
|---|---|---|
| B·R·A·H·M·S CgA II KRYPTOR | ARCHITECT CEA | |
| Intended Use andIndications for Use | B·R·A·H·M·S™ CgA IIKRYPTOR™ is an automatedimmunofluorescent assay usingTime-Resolved Amplified CryptateEmission (TRACE™) technologyfor quantitative determination ofChromogranin A concentration inhuman serum. | The ARCHITECT CEA assay is aChemiluminescent MicroparticleImmunoassay (CMIA) for thequantitative determination ofCarcinoembryonic Antigen (CEA)in human serum and plasma. |
| B·R·A·H·M·S™ CgA IIKRYPTOR™ is to be used inconjunction with other clinicalmethods as an aid in monitoring ofdisease progression during thecourse of disease and treatment inpatients with gastroentero-pancreatic neuroendocrine tumors(GEP-NETS, grade 1 and grade 2). | The ARCHITECT CEA assay is tobe used as an aid in the prognosisand management of cancerpatients in whom changingconcentrations of CEA areobserved. |
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| Item | Subject Device: | Predicate Device:K990774 |
|---|---|---|
| B-R-A-H-M-S CgA II KRYPTOR | ARCHITECT CEA | |
| Operating Principle(Technology) | Automated fluorescentimmunoassay using TRACE(Time-resolved amplified cryptateemission) technology. | Automated chemiluminescentmicroparticle immunoassay (CMIA) |
| Measured Analyte | Chromogranin A (CgA) | Carcinoembryonic Antigen (CEA) |
| Test Matrix | Serum | Serum or plasma |
| Methodology | Automated, quantitative | Same |
| Sample volume | 14 µL | 10 µL |
| Antibody | 2 antibodies labelled witha) cryptate (the donor, to beexcited by laser light) andb) fluorophore (the acceptor;fluorescence emission) | 2 antibodies labelled witha) paramagnetic microparticles(for immune complex formation)b) acridinium (chemiluminescentdye, signal emission) |
| Assay Principle | Sandwich Immunoassay | Same |
| Principal Operator | Professional user | Same |
| Calibrator (CAL) | 1 calibrator | 2 calibrators |
| Instrument / Analyzer | B-R-A-H-M-S KRYPTOR compactPLUS analyzer | ARCHITECT analyzer series |
6.2 Summary of Non-Clinical Test - Performance and Safety Testing 807.92(b)(1):
| Standards Body | Standard Name |
|---|---|
| Clinical LaboratoryStandards Institute(CLSI) | CLSI EP05-A3 – Evaluation of Precision of Quantitative MeasurementProcedures;Approved Guideline - Third Edition. |
| CLSI | CLSI EP06-Ed2 – Evaluation of the Linearity of Quantitative MeasurementProcedures: A Statistical Approach; Approved Guideline. |
| CLSI | CLSI EP07-A3 - Interference testing in clinical chemistry; approvedguideline - Third Edition |
| CLSI | CLSI EP09-A3 - Measurement Procedure Comparison And Bias EstimationUsing Patient Samples - 3rd Edition |
| CLSI | CLSI EP17 A2: - Evaluation of Detection Capability for Clinical LaboratoryMeasurement Procedures; Approved Guideline - Second Edition |
| CLSI | CLSI EP25 A:2009 - Evaluation of Stability of In Vitro Diagnostic Reagents;Approved Guideline |
| CLSI | CLSI EP28-A3c - Defining, Establishing, and Verifying Reference Intervalsin the Clinical Laboratory; Approved Guideline - Third Edition |
| CLSI | CLSI EP34 1st Edition - Establishing and Verifying an Extended MeasuringInterval Through Specimen Dilution and Spiking, 1st Edition |
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| Standards Body | Standard Name |
|---|---|
| InternationalOrganization forStandardization (ISO) | ISO 14971:2019 Medical Devices - Application of Risk Management toMedical Devices |
| ISO | ISO 15223-1: 2016 Medical Devices – Symbols to be used with MedicalDevice Labels, Labelling and Information to be Supplied Part 1: GeneralRequirements |
| ISO | ISO 17511:2020 In Vitro diagnostic medical devices - Measurement ofquantities in biological samples - Metrological traceability of valuesassigned to calibrators and control materials |
8. Test Principle
The B-R-A-H-M-S CgA II KRYPTOR assay is a one-step immunoassay to determine the presence of CgA in human serum using TRACE™ technology. One reagent antibody is labelled with Alexa Fluor™ 647 (the acceptor). The second antibody is labelled with cryptate (the donor). After excitation with laser light, spatial proximity of the two labelled antibodies in the antigen/antibody complex allows for energy transfer from the donor to the acceptor molecule and subsequent fluorescent signal emission as relative light units (RLUs) which can be detected by the B-R-A-H-M-S KRYPTOR instrument. A direct relationship exists between the amount of CgA in the sample and the RLUs detected by the B-R-A-H-M-S KRYPTOR instrument's optical system.
9. Performance Characteristics
a. Analytical Performance
Repeatability and Within-Laboratory Precision i.
Precision (repeatability, within-laboratory, reproducibility and lot-to-lot precision) was determined following CLSI Guideline EP05-A3.
Repeatability and Within-Laboratory coefficient of variation (CV) were calculated from the measurement of samples over 20 days, with 2 runs per day in 2 replicates:
| Sample IDN = 12 | Mean Value (ng/mL) | Repeatability CV | Within-Laboratory CV |
|---|---|---|---|
| P03 | 23.0 | 5.2 % | 10.0 % |
| P04 | 26.0 | 5.1 % | 9.9 % |
| P05 | 34.6 | 3.7 % | 8.8 % |
| P06 | 56.3 | 2.8 % | 8.4 % |
| P07 | 86.3 | 1.7 % | 8.3 % |
| P08 | 132 | 1.3 % | 7.1 % |
| P09 | 228 | 1.7 % | 6.9 % |
| P10 | 463 | 1.1 % | 4.4 % |
| 39PRE05 | 744 | 1.6% | 2.8 % |
| P11 | 1,177 | 1.1 % | 4.0 % |
| P12 | 1,825 | 1.3 % | 4.7 % |
| P13 | 2,687 | 1.6 % | 7.4 % |
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Lot-to-lot CVs were calculated from the measurement of samples over 5 days, with 5 replicates per day using 3 reagent lots and 1 instrument:
| Sample | Mean Value (ng/mL) | Lot-to-Lot | |
|---|---|---|---|
| SD | CV | ||
| 39LOT_01 | 26,3 | 0.3 | 1.2% |
| 39LOT_02 | 48,9 | 0.0 | 0.0% |
| 39LOT_03 | 96,8 | 0.1 | 0.1% |
| 39LOT_04 | 513 | 8.3 | 1.6% |
| 39LOT_05 | 757 | 14.0 | 1.9% |
| 39LOT_06 | 963 | 23.6 | 2.4% |
| 39LOT_07 | 1,441 | 39.1 | 2.7% |
| 39LOT_08 | 2,030 | 88.9 | 4.4% |
| 39LOT_09 | 2,540 | 19.2 | 0.8% |
| 39LOT_10 | 2,895 | 0.0 | 0.0% |
ii. Reproducibility
For reproducibility, samples have been measured on 5 days, with 2 runs per day in 3 replicates using 1 reagent lot at 3 different sites (different instruments).
| Sample | [CgA]Mean(ng/mL) | Repeatability | Between-Run | Between-Day | Between-Site | Reproducibility | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| No. | SD | CV | SD | CV | SD | CV | SD | CV | SD | CV | |
| 2.1 | 25.7 | 1.66 | 6.5% | 0.0 | 0.0% | 0.55 | 2.2% | 1.50 | 5.8% | 2.30 | 9.0% |
| 2.2 | 53.6 | 1.93 | 3.6% | 1.53 | 2.8% | 2.94 | 5.5% | 0.00 | 0.0% | 3.83 | 7.1% |
| 2.3 | 104 | 2.07 | 2.0% | 1.51 | 1.5% | 2.04 | 2.0% | 2.88 | 2.8% | 4.37 | 4.2% |
| 2.4 | 515 | 9.52 | 1.8% | 5.87 | 1.1% | 9.55 | 1.9% | 12.6 | 2.5% | 19.4 | 3.8% |
| 2.5 | 1,089 | 16.7 | 1.5% | 23.9 | 2.2% | 41.0 | 3.8% | 38.9 | 3.6% | 63.6 | 5.8% |
| 2.6 | 1,723 | 29.2 | 1.7% | 63.4 | 3.7% | 61.8 | 3.6% | 0.0 | 0.0% | 93.2 | 5.4% |
| 2.7 | 2,923 | 62.3 | 2.1% | 66.8 | 2.3% | 64.5 | 2.2% | 178 | 6.1% | 210 | 7.2% |
| 2.8 | 92,561 | 2,669 | 2.9% | 2,993 | 3.2% | 1,584 | 1.7% | 2,803 | 3.0% | 5,143 | 5.6% |
iii. Detection Capability
The LoB (Limit of Blank), LoD (Limit of Detection) and LoQ (Limit of Quantitation) of the assay were determined based on the guidelines from CLSI EP17-A02.
a. LoB Test Protocol
LoB was determined by testing a total of 30 replicates of 4 different blank samples (120 total replicates) over 8 days with 2 reagents lots on 2 B-R-A-H-M-S KRYPTOR compact PLUS instruments. The LoB was determined to be 11.3 ng/mL, using the parametric option.
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b. LoD Test Protocol
LoD was determined by testing a total of 30 replicates of 4 different low CgA concentration samples (120 total replicates) over 7 days with 2 reagents lots on 2 B-R-A-H-M-S KRYPTOR compact PLUS instruments. The LoD was determined to be 14.0 ng/mL. using the parametric option.
c. LoQ Test Protocol
LoQ was determined by testing a total of 60 replicates of 7different pools of human serum samples collected from healthy individuals (420 total replicates) over 5 days with 3 reagents lots on 3 B-R-A-H-M-S KRYPTOR compact PLUS instruments. LoQ was defined as the lowest concentration that can be reproducibly measured with an intermediate within-laboratory precision CV of ≤ 20%. The LoQ was determined to be 20.0 ng/mL.
iv. Linearity
Linearity was determined following CLSI Guideline EP06-A on study design and revised CLSI EP06-A 2nd Edition guidance for analysis.
The B.R.A.H.M.S C.gA II KRYPTOR assay is linear from 20.0 ng/mL (LoQ) up to 1,000,000 ng/mL. Samples above 3,000 ng/mL will be diluted automatically.
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| Sample | Range (ng/mL) | Slope(95%CI) | Intercept(95%CI) | R2 |
|---|---|---|---|---|
| Sample 01 | 296-3401 | 1.021(1.009;1.033) | -66.11(-73.07;-59.15) | 0.998 |
| Sample 02 | 39-301 | 1.081(1.065;1.096) | -10.96(-12.77;-9.150) | 0.997 |
| Sample 03 | 15-52 | 1.036(1.012;1.061) | -2.048(-2.853;-1.243) | 0.994 |
| Sample 01-02-03(combined data) | 15-3401 | 0.975(0.961;0.990) | -0.447(-1.407;0.514) | 0.992 |
Dilution Recovery V.
Dilution recovery was determined by diluting 10 pools of human serum samples. Dilutions were done by B-R-A-H-M-S KRYPTOR compact PLUS instruments using the kit diluent. The recoveries between the pure values and each dilution level values and mean recoveries were calculated. The mean recovery values are ranging from 97.6% - 109.6%.
Spike Recovery vi.
A total of 13 individual serum samples with different CgA concentrations were used to assess spike recovery of the B-R-A-H-M-S CgA II KRYPTOR. 7 of the samples were spiked (1:1) with a low concentration recombinant CgA antigen sample and the 6 other samples were spiked with a second recombinant CgA antigen sample of higher concentration. The samples were measured (in duplicate) before and after spiking and the recoveries between the measured mean values and the expected concentrations were calculated for the 13 spiked pools. The spiked pools had individual recovery values ranging from 91% - 109%.
High Dose Hook Effect vii.
B-R-A-H-M-S CgA II KRYPTOR is a homogenous immunoassay and does not require separation or washing steps. It is thus possible to obtain data without interrupting the immunological reaction. High concentration samples (> 3,000 ng/mL) are detected in the first few seconds of incubation and may be diluted by the appropriate dilution factor, then re-assayed automatically.
- In other words, potential Hook Effect is detected by kinetics analysis of the samples by ● B-R-A-H-M-S KRYPTOR analyzer family. Measurement is stopped for samples greater than 3.000 ng/mL. If automatic dilution is activated, then the B-R-A-H-M-S KRYPTOR analyzer automatically dilutes the sample at an appropriate dilution. If automatic dilution is not activated, then the B-R-A-H-M-S KRYPTOR analyzer family adds the dilution of the sample to the worklist and the user has to validate the worklist to launch the dilution of the sample. This process allows for sample measurements greater than 3,000 ng/mL up to 1,000,000 ng/mL.
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Interference and Cross-Reactivity viiii.
Interference studies were based on CLSI EP07-A3 as a guideline. Cross-reactivity studies were based on CLSI EP07-A2 as a guideline and were verified for compliance with EP07-A3.
Interference was assessed in accordance with CLSI Guideline EP7-A3 and CLSI EP37 1* edition. The substances evaluated with the B-R-A-H-M-S CgA II KRYPTOR were found not to affect the test performance (bias ≤ 10%) at concentrations reasonably and consistently found in clinical situations. The substances included the following:
| Endogenous Interfering Substance | Concentration |
|---|---|
| Hemoglobin | 10 g/L |
| Bilirubin (unconjugated) | 500 mg/L |
| Triglycerides | 5 g/L |
| Albumin | 50 g/L |
| HAMAs | 300 μg/L |
| Rheumatoid factors | 1,000 kIU/L |
| Exogenous Interfering Substance | Concentration |
|---|---|
| Acetaminophen | 238.3 mg/L |
| Alprazolam | 6.0 mg/L |
| Amlodipine | 100.2 µg/L |
| Aspirin | 546.6 mg/L |
| Biotin | 3,510 ng/mL |
| Fish Oil | 2.4 g/L |
| Hydrochlorothiazide | 6.0 mg/L |
| Ibuprofen | 499.6 mg/L |
| Lisinopril | 300.4 µg/L |
| Lorazepam | 998.3 µg/L |
| Metoprolol | 5.0 mg/L |
| Multivitamin:Vitamin A | 16.7 kIU/L |
| Vitamin C | 1,000 mg/L |
| Vitamin D | 5.33 kIU/L |
| Vitamin E | 100.0 IU/L |
| Thiamin (B1) | 200 mg/L |
| Riboflavin (B2) | 250 mg/L |
| Niacin | 170 mg/L |
| Vitamin B6 | 170 mg/L |
| Vitamin B12 | 3,330 µg/L |
| Bevacizumab | 720 mg/L |
| Capecitabine | 2.85 g/L |
| Carboplatin | 1 g/L |
| Cisplatin | 2 g/L |
| Etoposide | 114 mg/L |
| Everolimus | 6 mg/L |
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| Exogenous Interfering Substance | Concentration |
|---|---|
| Fluorouracil | 684 mg/L |
| Interferon (IFN-α-2b) | 3,000 kU/L |
| Lanreotide | 72 mg/L |
| Octreotide | 12 mg/L |
| Oxaliplatin | 96.9 mg/L |
| Sunitinib | 22.5 mg/L |
| Temozolomide | 228 mg/L |
| Temsirolimus | 15 mg/L |
| Pancrelipase | 480 kU/L |
| Hydrocodone | 200.3 µg/L |
| Oxycodone | 500.9 μg/L |
Cross-reactivity effects were tested with substances that are structurally similar to the CgA protein. The percent of cross-reactivity was between -21.6% - 0.03%. The results are shown below.
| Potentially Cross-Reacting Molecule | Concentration |
|---|---|
| Parastatin (porcine) | 100 nmol/L |
| Catestatin (human) | 452 nmol/L |
| Pancreastatin (human) | 182 nmol/L |
| Vasostatin I (human) | 9 nmol/L |
| Vasostatin II Cterm (human) | 15 nmol/L |
| Vasostatin II (human) | 5 nmol/L |
| Chromostatin (bovin) | 10 nmol/L |
| Chromogranin A protein fragment (human) | 217 nmol/L |
| Chromogranin B (Secretogranin 1) (human) | 72 nmol/L |
| Chromogranin C (Secretogranin 2) (human) | 148 nmol/L |
| WE14 (human) | 606 nmol/L |
Sample Stability İX.
The following studies were performed to confirm the sample stability claims.
a. Short-Term Sample Stability at Room Temperature
- · A study with 12 serum samples stored at room temperature (18-25°C) confirms a stability claim of 48 hours for storage at room temperature.
b. Long-Term Sample Stability at -20°C
- A total of 4 serum samples, each divided into 10 aliquots, were tested after storage at -20°C to demonstrate long-term stability. The study confirms a stability claim of 1 month for storage at -20°C.
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c. Freeze Thaw Sample Stability
- · A total of 28 serum samples were tested in two studies to demonstrate freeze-thaw stability. The studies confirm a stability claim of 4 freeze-thaw cycles (storage at -20℃).
Reagent Stability x.
The following studies were conducted to support reagent stability claims based on CLSI EP25-A.
a. Real Time Stability for B-R-A-H-M-S CgA II KRYPTOR
Real time stability study with native samples for 1 lot of unopened B-R-A-H-M-S CgA II KRYPTOR reagent stored at 2-8°C has been carried out for 4 months. In combination with an accelerated stability study, stability for reagents at 2-8°C is supported for a duration of 9 months. An additional real-time stability study conducted on three B-R-A-H-M-S CgA II KRYPTOR reagent lots using human serum samples for > 9 months storage claims is ongoing.
b. Real Time Stability for B-R-A-H-M-S CqA II KRYPTOR CAL
- Real time stability study for 3 lots of unopened B-R-A-H-M-S CgA II KRYPTOR calibrators stored at 2-8°C has been carried out for up to 112 weeks. Real time stability for calibrators at 2-8°C is claimed at 104 weeks (24 months).
c. Real Time Stability for B-R-A-H-M-S CgA II KRYPTOR QC
- Real time stability study for 3 lots of unopened B-R-A-H-M-S CgA II KRYPTOR controls stored at 2-8°C has been carried out for up to 109 weeks at least. Real time stability for controls at 2-8°C is claimed at 104 weeks (24 months).
d. On-board Stability for B-R-A-H-M-S CgA II KRYPTOR Assay
- In-use stability study for 1 lot of opened and reconstituted B-R-A-H-M-S CgA II KRYPTOR reagents stored at 2-8°C on board a B-R-A-H-M-S KRYPTOR compact PLUS instrument has been carried out for up to 30 days. In-use stability for B-R-A-H-M-S CgA II KRYPTOR reagents stored at 2-8°C is claimed at 29 days, with a required calibration after 15 days.
e. On-board Stability for B-R-A-H-M-S CgA II KRYPTOR CAL
- · In-use stability study for 1 lot of opened and reconstituted B-R-A-H-M-S CgA II KRYPTOR calibrator stored at room temperature (18-25°C) on board a B-R-A-H-M-S KRYPTOR compact PLUS instrument has been carried out for up to 6 hours. In-use stability for B-R-A-H-M-S CaA II KRYPTOR calibrators stored at room temperature is claimed at 5 hours.
f. On-board Stability for B-R-A-H-M-S CgA II KRYPTOR QC
- In-use stability study for 1 lot of opened and reconstituted B-R-A-H-M-S CgA II KRYPTOR controls stored at room temperature (18-25°C) on board a B·R·A·H·M·S KRYPTOR compact PLUS instrument has been carried out for up to 6 hours. In-use stability for B-R-A-H-M-S CgA II KRYPTOR controls stored at room temperature is claimed at 5 hours.
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- In-use stability study for 1 lot of opened and reconstituted B-R-A-H-M-S CgA II KRYPTOR controls stored at 2-8°C has been carried out for up to 29 hours. In-use stability for B-R-A-H-M-S CqA II KRYPTOR controls stored at 2-8°C is claimed at 24 hours.
- · In-use stability study for 1 lot of opened and reconstituted B-R-A-H-M-S CgA II KRYPTOR controls stored at ≤ -20℃ has been carried out for up to 36 days. In-use stability for B-R-A-H-M-S CgA II KRYPTOR controls stored at ≤ -20°C is claimed at 31 days.
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i. Clinical Cut-off:
The clinical cut-off was derived from a retrospective, bicentric observational pilot study of 102 patients with diagnosed well-differentiated G1 and G2 GEP-NETs in the US. During routine monitoring visits serum CgA concentrations were assessed in comparison to standard imaging (CT/MRI) and tumors were classified by RECIST 1.1 criteria for progression (progressive disease) vs. no progression (complete response, partial response or stable disease).
ΔCgA > 50% and CgA > 100 ng/ml:
An increase of CgA serum concentrations of more than 50% to a value of greater than 100 ng/ml between consecutive monitoring visits defines a positive test result representing a higher probability that a tumor progression has occurred
ΔCgA ≤ 50% or CgA ≤ 100 ng/ml:
A change of CgA serum concentrations of equal or less than 50% increase between monitoring visits or to a value of 100 ng/ml or less defines a negative test result representing a lower probability that a tumor progression has occurred
ii. Clinical Sensitivity and Specificity:
The prospective study validated a statistically significant association of a 50% increase of CgA serum concentrations to a value of greater than 100 ng/ml between consecutive monitoring visits in 153 adult GEP-NET patients (grade 1 and 2) with tumor progression as classified by RECIST 1.1 criteria. The study vielded (estimate and 95% confidence interval):
| Tumor Progression | ||||
|---|---|---|---|---|
| Progression | No Progression | Total | ||
| Binary CgAincrease* | Positive | 33 | 24 | 57 |
| Negative | 63 | 339 | 402 | |
| Total | 96 | 363 | 459 |
*Cut-off 50% CgA increase and CgA > 100 ng/mL
| PerformanceMeasurement | Value | LowerCl* | UpperCl* |
|---|---|---|---|
| Sensitivity | 34.4% (33/96) | 23.2% | 45.5% |
| Specificity | 93.4% (339/363) | 90.2% | 96.0% |
| PPV | 57.9% (33/57) | 40.5% | 73.6% |
| NPV | 84.3% (339/402) | 79.3% | 89.1% |
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| PerformanceMeasurement | Value | LowerCl* | UpperCl* |
|---|---|---|---|
| PLR | 5.20 | 2.90 | 9.66 |
| NLR | 0.70 | 0.58 | 0.83 |
| Prevalence | 20.9% (96/459) |
*CI = 95% Confidence interval
PPV = positive predictive value; NPV = negative predictive value PLR = positive likelihood ratio; NLR = negative likelihood ratio
PLR = positive likelihood ratio; NLR = negative likelihood ratio
iii. Expected Values/Reference Ranges:
The B-R-A-H-M-S CgA II KRYPTOR reference limit was determined by testing a total of 206 samples from self-declared healthy individuals in the USA. The 95th quantile was estimated 187.0 ng/mL (90% confidence interval 123.5 to 255.6 ng/mL).
10. Proposed Labeling
The labeling is sufficient, and it satisfies the requirements of 21 CFR Part 809.10.
11. Conclusion
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
§ 866.6010 Tumor-associated antigen immunological test system.
(a)
Identification. A tumor-associated antigen immunological test system is a device that consists of reagents used to qualitatively or quantitatively measure, by immunochemical techniques, tumor-associated antigens in serum, plasma, urine, or other body fluids. This device is intended as an aid in monitoring patients for disease progress or response to therapy or for the detection of recurrent or residual disease.(b)
Classification. Class II (special controls). Tumor markers must comply with the following special controls: (1) A guidance document entitled “Guidance Document for the Submission of Tumor Associated Antigen Premarket Notifications (510(k)s) to FDA,” and (2) voluntary assay performance standards issued by the National Committee on Clinical Laboratory Standards.