(765 days)
Not Found
No
The device description and performance studies focus on a standard real-time PCR assay and its analytical and clinical performance metrics, with no mention of AI or ML algorithms for data analysis or interpretation.
No
This device is for screening newborns for certain conditions, not for treatment.
No
The text explicitly states: "This test is not intended for use as a diagnostic test and a positive screening result should be followed by confirmatory testing."
No
The device is described as a "multiplex real-time PCR-based assay" and includes "reagents" as part of the kit. This indicates it is a physical kit with chemical components, not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states that the kit is for the "semi-quantitative determination of TREC... as an aid in screening newborns for Severe Combined Immunodeficiency (SCID) and for the semi-quantitative determination of KREC... as an aid in screening newborns for X-linked agammaglobulinemia (XLA)." This describes a test performed in vitro (outside the body) on a biological specimen (blood) to provide information about a patient's health status (screening for SCID and XLA).
- Device Description: The description details a "multiplex real-time PCR-based assay" that uses reagents to amplify and detect targets in DNA extracted from blood specimens. This is a typical description of an in vitro diagnostic test kit.
- Anatomical Site: The test is performed on "Blood specimens dried on a filter paper," which is a biological sample taken from the body.
- Intended User / Care Setting: The intended users are "Newborn screening laboratories," which are facilities where in vitro diagnostic testing is performed.
- Performance Studies: The document includes detailed performance studies (Site-to-Site Reproducibility, Precision, Limit of Detection, Linearity, Interference, Screening Performance) with metrics like Sensitivity and Specificity. These are standard evaluations for in vitro diagnostic devices to demonstrate their analytical and clinical performance.
- Predicate Device: The mention of a "Predicate Device" (PerkinElmer ENLITE™ Neonatal TREC Kit) with a K number (DEN140010) strongly indicates that this device is being compared to a previously cleared IVD.
All of these factors align with the definition and characteristics of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The Eonis™ SCID-SMA kit is intended for the semi-quantitative determination of TREC (T-cell receptor excision circle) as an aid in screening newborns for Severe Combined Immunodeficiency (SCID) and for the semi-quantitative determination of KREC (Kappa-deleting recombination excision circle) as an aid in screening newborns for X-linked agammaglobulinemia (XLA). The test is intended for DNA from blood specimens dried on a filter paper and for use on the QuantStudio™ Dx Real-Time PCR instrument.
This test is not intended for screening of SCID-like Syndromes, such as DiGeorge Syndrome, It is also not intended to screen for less acute SCID syndromes such as leaky-SCID or variant SCID. The test is not indicated for screening B-cell deficiency disorders other than XLA, such as atypical XLA, or for screening of XLA carriers.
This test is not intended for use as a diagnostic test and a positive screening result should be followed by confirmatory testing.
Product codes
PJI
Device Description
The Eonis SCID-SMA kit is a multiplex real-time PCR-based assay. It uses target sequence-specific primers and TaqMan™ probes to amplify and detect three targets: TREC, and RPP30, in the DNA extracted from newborn dried blood spot (DBS) using Eonis DNA Extraction kit in a single PCR reaction.
Each Eonis SCID-SMA kit contains reagents for up to 384 reactions (for 3241-001U) or 1152 reactions (for 3242-001U) including kit controls.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
Newborns
Intended User / Care Setting
Not Found
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
The screening performance of the Eonis SCID-SMA kit was determined in a clinical study conducted in Denmark. Retrospective archived dried blood spot specimens (collected from US and Denmark) from subjects confirmed positive for SCID, XLA were included to enrich the cohort of routine newborn screening specimens obtained from the Danish Newborn Screening Biobank.
Confirmatory test results were used as the comparator for the confirmed positive SCID, XLA cases. The clinical status of the routine subjects was determined through a retrospective review by clinical experts to confirm the routine subject cohort samples were from unaffected individuals.
Using the data collected to establish the expected values of the Eonis SCID-SMA kit were determined by calculating the TREC concentrations corresponding to the 0.3" and 1.0" population percentiles (262 copies/10 cells for TREC, 484 copies/10 cells for KREC) established in a cut-off study with an independent dataset. The specimens having TREC and KREC levels below the cut-off values in the initial round of testing were re-tested in duplicate. The final results (presumptive positive positive, invalid result) were classified after the second round of testing.
In total, 17 SCID and 6 XLA retrospective case specimens and 3018 normal newborn screening specimens were available for the study.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Screening Performance Study:
- Clinical study conducted in Denmark using retrospective archived dried blood spot specimens from US and Denmark.
- Sample size: 3018 clinical newborn samples with known medical status (17 confirmed positive for SCID and 6 confirmed positive for XLA).
- Sensitivity (TREC): 100%
- Specificity (TREC): 99.7%
- False-negative rate (TREC): 0%
- False-positive rate (TREC): 0.3%
- Sensitivity (KREC): 100%
- Specificity (KREC): 99.7%
- False-negative rate (KREC): 0%
- False-positive rate (KREC): 0.3% (for female population, 0.1%)
Site-to-site Reproducibility Study:
- Determined using a panel of dried blood spots at different TREC, KREC levels.
- 1 reagent kit lot, 2 external newborn screening laboratories, and 1 internal site.
- Each laboratory: 2 operators performed 5 runs each during 5 operating days.
- Each run consisted of 1 plate with 5 replicates per sample.
- Total number of measurements: 150 per sample in each laboratory.
- Showed various levels of lognormal %CV for TREC (27%-180%) and KREC (21%-89%) across different sample concentrations.
Precision Study:
- Determined in accordance with CLSI document EP05-A3.
- Used dried blood spot samples, 3 kit lots, 3 sets of Eonis test systems (including three JANUS Extraction Instruments, three JANUS PCR Mastermix Instruments and three QuantStudio™ Dx Real-Time PCR Instruments).
- 2 operators, and 54 runs over 23 calendar days.
- Each run consisted of 1 plate with 2 replicates per sample in a randomized plate map.
- Total number of measurements: 108 per sample.
- Showed various levels of lognormal %CV for TREC (27%-125%) and KREC (31%-178%) across different sample concentrations.
Limit of Detection Study:
- Determined in accordance with CLSI document EP17-A2.
- LoB for TREC, KREC, SMN1: 0 copies/μL blood and 0 copies/10^5 cells (based on 300 determinations of blank samples).
- LoD for TREC: 242 copies/10^5 cells with 95% probability (based on 960 determinations, 20 replicates per dilution).
- LoQ for TREC: 242 copies/10^5 cells (equal to LoD).
- LoD for KREC: 459 copies/10^5 cells with 95% probability.
- LoQ for KREC: 459 copies/10^5 cells (equal to LoD).
Linearity Study:
- Determined in accordance with CLSI document EP06 ED2:2020 with one kit lot.
- Three (3) sets of contrived samples were used, diluted to 9 levels and tested with 4 replicates for each level.
- TREC: Linear from 94 copies/10^5 cells with observed maximum deviation of -14.3%.
- KREC: Linear from 117 to 24343 copies/10^5 cells with observed maximum deviation of 17.3%.
Interference Study:
- Evaluated in accordance with CLSI document EP07-A3.
- Tested substances (Conjugated bilirubin, Hemoglobin, Unconjugated bilirubin, Intralipid®, Li-heparin, EDTA, Na-citrate) at specified concentrations were found not to interfere.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
TREC:
- Sensitivity: 100 %
- False-negative rate: 0 %
- Specificity: 99.7 %
- False-positive rate: 0.3 %
KREC:
- Sensitivity: 100 %
- False-negative rate: 0 %
- Specificity: 99.7 %
- False-positive rate: 0.3 %
KREC (Male only):
- False-positive rate: 0.5% (for male only population)
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
PerkinElmer ENLITE™ Neonatal TREC Kit (DEN140010)
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
Not Found
§ 866.5930 Newborn screening test for severe combined immunodeficiency disorder (SCID).
(a)
Identification. A newborn screening test for SCID is a prescription device intended to measure T-cell receptor excision circle (TREC) DNA obtained from dried blood spot specimens on filter paper using a polymerase chain reaction based test as an aid in screening newborns for SCID. Presumptive positive results must be followed up by diagnostic confirmatory testing. This test is not intended for use as a diagnostic test, or for screening of SCID-like syndromes, such as DiGeorge syndrome or Omenn syndrome. It is also not intended to screen for less acute SCID syndromes, such as leaky SCID or variant SCID.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information:
(i) The intended use must indicate:
(A) The test is not intended for diagnostic use, or for screening of SCID-like syndromes, such as DiGeorge syndrome or Omenn syndrome; and
(B) The test is not intended to screen for less acute SCID syndromes, such as leaky SCID or variant SCID.
(ii) A detailed description of all components in the test that includes:
(A) A detailed description of the test components, all required reagents, instrumentation and equipment, including illustrations or photographs of nonstandard equipment or methods;
(B) Detailed documentation of the device software including, but not limited to, standalone software applications and hardware-based devices that incorporate software;
(C) Specifications for the filter paper, which must be appropriately labeled for in vitro diagnostic use, to be used in specimen collection and how it will be used in specimen collection validation. These specifications must include: descriptive characteristics of the filter paper, instructions on how a lab should choose the appropriate filter paper, chemical properties of the filter paper, interference concerns associated with the chemicals in the filter paper, absorption properties of the filter paper, punch size, absorption capacity, testing for homogeneity of punches, diameter of the circle for the dried blood spot aliquot, absorption time, physical composition, and number and size of punches to be tested;
(D) Methodology and protocols for detection of T-cell receptor excision circles and methods for determination of results. The cutoff must be selected before conducting clinical and analytical studies;
(E) A description of the result outputs along with sample reports. Sample reports must include the scale used in reporting of results (
e.g., TREC copies/µL) and the range of values that will be reported out; and(F) A description of appropriate internal and external controls that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure.
(iii) Information that demonstrates the performance characteristics of the test, including:
(A) Data that demonstrates the clinical validity of the device, using well characterized prospectively or retrospectively obtained clinical specimens representative of the intended use population. A minimum of 10 to 15 confirmed positive specimens must be obtained from more than 1 site, including relevant annotation, and, at 1 year or beyond, a SCID diagnosis by flow cytometry or clinically meaningful information regarding the status of the subject must be obtained. Additional specimens should have been obtained that are characterized by other disorders that can be found by screening specimens that have low or absent TREC (
e.g., other T-cell lymphopenic disorders) to supplement the range of results. The clinical validation study must have a pre-specified clinical decision point (i.e., cutoff to distinguish positive and negative results). Results must be summarized in tabular format comparing interpretation of results to the reference method. Point estimates together with two-sided 95 percent confidence intervals must be provided for the positive percent agreement, negative percent agreement, and overall percent agreement. Data must include the retest rate, the false positive rate before retest, the final false positive rate, and the false negative rate;(B) Device reproducibility data generated, using a minimum of three sites of which at least two must be external sites, with two operators at each site. Each site must conduct a minimum of five runs per operator over five nonconsecutive days evaluating a minimum of six different relevant TREC concentrations that span and are well distributed over the measuring range and include the clinical cutoff. Specimens must include cord blood and cord blood diluted with ABO matched adult blood specimens. Identical specimens from the same sample panel must be tested at each site. Each specimen must be run in triplicate and include controls run in triplicate. Results must be reported as the standard deviation and percentage coefficient of variation for each level tested. Results must also be displayed as a dichotomous variable around the cutoff. Total variation must be partitioned into the sum of within-lab and between-lab variations with pre-specified acceptance criteria and 95 percent confidence intervals for all data. Pre-specified acceptance criteria must be provided and followed;
(C) Device precision data using clinical samples to evaluate the within-lot, between-lot, within-run, between run, and total variation. A range of TREC levels of the specimen must include samples within the measuring range, samples above and below the measuring range, as well as with samples very near above and below the cutoff value. At least three replicates of each specimen must be tested with controls and calibrator(s) according to the device instructions for use. The precision study must use well characterized samples using different lots, instruments, and operators. Results must be summarized in tabular format. Pre-specified acceptance criteria must be provided and followed;
(D) Linearity of the test must be demonstrated using a dilution panel from clinical samples. The range of dilution samples must include samples within the measuring range, samples above and below the measuring range, as well as with samples very near above and below the cutoff value. Results of the regression analysis must be summarized in tabular format and fitted into a linear regression model with the individual measurement results against the dilution factors. Pre-specified acceptance criteria must be provided and followed;
(E) Device analytic sensitivity data, including limit of blank, limit of detection, and limit of quantification;
(F) Device specificity data, including interference, carryover, cross-contamination, and in silico analysis of potential off-target genomic sequences;
(G) Device stability data, including real-time stability of samples under various storage times, temperatures, and freeze-thaw conditions. A separate shipping stability study must be performed;
(H) Lot-to-lot reproducibility study of each filter paper that will be validated with the test. The lot-to-lot study must include a minimum of three lots of each blood spot card that will be validated with the test and be conducted over five nonconsecutive days. The sample panel must consist of specimens with a range of TREC levels and include samples within the measuring range, samples above and below the measuring range, and samples very near above and below the cutoff value. Multiple punches must be obtained from each card for demonstration of homogeneity of the analyte across the dried blood spot. Comparability of the test performance for each filter paper must be demonstrated. Stability and storage of TREC DNA on each blood spot card must be demonstrated. Results of the lot-to-lot study must be summarized providing the mean, standard deviation, and percentage coefficient of variation in a tabular format. Data must be calculated for within-run, between-run, within-lot, and between-lot. Data demonstrating the concordance between results across different filter papers must be provided. Study acceptance criteria must be provided and followed; and
(I) If applicable, a thermocycler reproducibility study must be performed using thermocyclers from three independent thermocyler manufacturers. The sample panel must consist of specimens with a range of TREC levels and must include samples within the measuring range, samples above and below the measuring range, and samples very near above and below the cutoff value. The study must be done using three filter paper lots and conducted over five nonconsecutive days. Results of the thermocycler reproducibility study must be summarized providing the mean, standard deviation, and percentage coefficient of variance in a tabular format. Data must be calculated for the within-run, between-run, within-lot, between-lot, and between thermocycler manufacturer study results. Study acceptance criteria must be provided and followed.
(iv) Identification of risk mitigation elements used by your device, including a description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing.
(2) Your § 809.10 compliant labeling must include:
(i) A warning statement that reads “This test is not intended for diagnostic use, preimplantation or prenatal testing, or for screening of SCID-like syndromes, such as DiGeorge syndrome or Omenn syndrome. It is also not intended to screen for less acute SCID syndromes, such as leaky SCID or variant SCID.”;
(ii) A warning statement that reads “Test results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods and clinical evaluation, as appropriate.”;
(iii) A description of the performance studies listed in paragraph (b)(1)(iii) and a summary of the results; and
(iv) A description of the filter paper specifications required for the test.
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November 9, 2022
PerkinElmer Inc Casey Fox, Ph.D. Sr. Manager, Regulatory Affairs 940 Winter St Waltham, MA 02451
Re: K203035
Trade/Device Name: Eonis SCID-SMA kit Regulation Number: 21 CFR 866.5930 Regulation Name: Newborn screening test for severe combined immunodeficiency disorder (SCID) Regulatory Class: Class II Product Code: PJI Dated: April 28, 2021 Received: April 30, 2021
Dear Dr. Fox:
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.
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
1
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).
Sincerelv.
Image /page/1/Picture/6 description: The image contains the name "Pamela S. Gallagher" in a large font. Next to the name is "-S" in a smaller font. On the right side of the image, there is the word "Digitally" followed by "S. Gallagher" and "Date: 20" and "-05'00'".
Digitally signed by Pamela S. Gallagher -S Date: 2022.11.09 09:36:15
Pamela Ebrahimi, Ph.D. Deputy Branch Chief Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health
Enclosure
2
Indications for Use
510(k) Number (if known) K203035
Device Name Eonis™ SCID-SMA kit
Indications for Use (Describe)
The Eonis™ SCID-SMA kit is intended for the semi-quantitative determination of TREC (T-cell receptor excision circle) as an aid in screening newborns for Severe Combined Immunodeficiency (SCID) and for the semi-quantitative determination of KREC (Kappa-deleting recombination excision circle) as an aid in screeming newborns for X-linked agammaglobulinemia (XLA). The test is intended for DNA from blood specimens dried on a filter paper and for use on the QuantStudio™ Dx Real-Time PCR instrument.
This test is not intended for screening of SCID-like Syndromes, such as DiGeorge Syndrome, It is also not intended to screen for less acute SCID syndromes such as leaky-SCID or variant SCID. The test is not indicated for screening B-cell deficiency disorders other than XLA, such as atypical XLA, or for screening of XLA carriers.
This test is not intended for use as a diagnostic test and a positive screening result should be followed by confirmatory testing.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| ☑ Prescription Use (Part 21 CFR 801 Subpart D) | ☐ Over-The-Counter Use (21 CFR 801 Subpart C) |
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510(k) Summary
This summary of safety and effectiveness information is supplied in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.
The assigned number is _k203035
| Submitted by: | PerkinElmer, Inc.
940 Winter Street
Waltham MA 02451 |
|-------------------|------------------------------------------------------------|
| Contact Person: | Casey Fox
Tel: 408-823-5561 |
| Trade Name: | Eonis SCID-SMA kit |
| Common Name: | Eonis SCID-SMA kit |
| Regulation: | 21 CFR 866.5930 |
| Classification: | II |
| Panel: | 75 Chemistry |
| Product Code: | PJI |
| Predicate device: | PerkinElmer ENLITE™ Neonatal TREC Kit (DEN140010) |
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1. Device Description
The Eonis SCID-SMA kit is a multiplex real-time PCR-based assay. It uses target sequence-specific primers and TaqMan™ probes to amplify and detect three targets: TREC, and RPP30, in the DNA extracted from newborn dried blood spot (DBS) using Eonis DNA Extraction kit in a single PCR reaction.
Each Eonis SCID-SMA kit contains reagents for up to 384 reactions (for 3241-001U) or 1152 reactions (for 3242-001U) including kit controls.
Table 1 Eonis SCID-SMA Kit content
| Component | Quantity |
|---|---|
| SCID-SMA Kit Controls | 2 filter paper cassettes containing 4 sets of dried blood |
| spots for 384 reaction kit | |
| 4 filter paper cassettes containing 8 sets of dried blood | |
| spots for 1152 reaction kit | |
| C1 Analyte-negative (TREC/KREC) control | |
| C2 Low TREC/KREC control | |
| C3 High TREC/KREC control | |
| PCR Reagent 1 | 1 vial, 2.7 mL for 384 reaction kit |
| 3 vials, 2.7 mL for 1152 reaction kit | |
| PCR Reagent 2 | 1 vial, 2.7 mL for 384 reaction kit |
| 3 vials, 2.7 mL for 1152 reaction kit | |
| Lot-specific | |
| quality control certificate | 1 pc |
2. Intended Use
The Eonis™ SCID-SMA kit is intended for the semi-quantitative determination of TREC (T-cell receptor excision circle) as an aid in screening newborns for Severe Combined Immunodeficiency (SCID) and for the semiquantitative determination of KREC (Kappa-deleting recombination excision circle) as an aid in screening newborns for X-linked agammaglobulinemia (XLA). The test is intended for DNA from blood specimens dried on a filter paper and for use on the QuantStudio™ Dx Real-Time PCR instrument.
This test is not intended for screening of SCID-like Syndromes, such as DiGeorge Syndrome, or Omenn Syndrome. lt is also not intended to screen for less acute SCID syndromes such as leaky-SCID or variant SCID. The test is not indicated for screening B-cell deficiency disorders other than XLA, such as atypical XLA, or for screening of XLA carriers.
TaqMan is a trademark of Roche Molecular Systems, Inc. QuantStudio is a trademark of Thermo Fisher Scientific.
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This test is not intended for use as a diagnostic test and a positive screening result should be followed by confirmatory testing.
3. Substantial Equivalency
The PerkinElmer Eonis SCID-SMA kit claims substantial equivalency to the PerkinElmer ENLITE™ Neonatal TREC Kit (DEN140010). Both devices are test systems intended for screening newborns for inherited immunodeficiency disorders.
The EnLite™ Neonatal TREC Kit is an in vitro diagnostic device intended for the semi-quantitative determination of TREC (T-cell receptor excision circle) DNA in blood specimens dried on filter paper. The test is indicated for use as an aid in screening newborns for severe combined immunodeficiency disorder (SCID).
Both devices are test systems intended for the semi-quantitative determination of TREC (T-cell receptor excision circle) DNA in blood specimens dried on filter paper on PCR instruments. In addition, Eonis SCID-SMA kit is intended for the semi-quantitation of KREC (Kappa-deleting recombination excision circle) as an aid in screening newborns for X-linked agammaglobulinemia (XLA).
The EnLite™ Neonatal TREC Kit uses a polymerase chain reaction (PCR) based nucleic acid amplification and time-resolved fluorescent-based detection to semi-quantify concentration. The PerkinElmer Eonis SCID-SMA Kit also uses fluorescent-based PCR nucleic acid amplification and detection to semi-quantify the same concentration. The use of PCR in newborn screening is well-established, and introduction of well-established real-time detection (qPCR) does not raise new questions of safety or effectiveness due to its use in this device.
Both devices are not intended for use as a diagnostic test or for screening of SCID-like Syndromes, such as DiGeorge Syndrome, or Omenn Syndrome. It is also not intended to screen for less acute SCD syndromes such as leaky-SCID or variant SCID.
In a study of 3018 clinical newborn samples with known medical status, 17 of which were confirmed positive for SCID and 6 were confirmed positive for XLA, the PerkinElmer Eonis SCID-SMA kit demonstrated the following sensitivity and specificity:
| Analyte | | Sensitivity | False-negative
rate | Specificity | False-positive
rate |
|---------|-------------------|-------------|------------------------|-----------------|------------------------|
| TREC | Percent | 100 % | 0 % | 99.7 % | 0.3 % |
| | Confidence Limits | 80.5 % - NA | NA - 19.5 % | 99.4 % - 99.9 % | 0.1 % - 0.6 % |
| KREC | Percent | 100 % | 0 % | 99.7 % | 0.3 % |
| | Confidence Limits | 54.1 % - NA | NA - 45.9 % | 99.4 % - 99.9 % | 0.1 % - 0.6 % |
In a study of 5437 clinical newborn samples with known medical status, 17 of which were confirmed positive for SCID, the PerkinElmer EnLite Neonatal TREC kit (excluding invalid results) demonstrated the following
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sensitivity and specificity (DEN140010):
| Analyte | | Sensitivity | False-
negative rate | Specificity | False-positive
rate |
|---------|-------------------|-------------|-------------------------|-----------------|------------------------|
| TREC | Percent | 100 % | 0 % | 99.7 % | 0.3 % |
| | Confidence Limits | 79.4 % - NA | NA - 20.6 % | 99.4 % - 99.8 % | 0.2 % - 0.6 % |
Table below provides the similarities and differences of the proposed device and the predicate. The analytical and clinical tests performed with the EONIS SCID-SMA kit have demonstrated that the two products are substantially equivalent.
| Characteristics | Proposed Device
EONIS SCID-SMA kit | Predicate
ENLITE™ Neonatal TREC Kit
(DEN140010). |
|-----------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use/
Indications for
Use | The Eonis™ SCID-SMA kit is intended for the semi-
quantitative determination of TREC (T-cell receptor
excision circle) as an aid in screening newborns for
Severe Combined Immunodeficiency (SCID) and for
the semi-quantitative determination of KREC
(Kappa-deleting recombination excision circle) as
an aid in screening newborns for X-linked
agammaglobulinemia (XLA). The test is intended for
DNA from blood specimens dried on a filter paper
and for use on the QuantStudio™ Dx Real-Time PCR
instrument.
This test is not intended for screening of SCID-like
Syndromes, such as DiGeorge Syndrome, or Omenn
Syndrome. It is also not intended to screen for less
acute SCID syndromes such as leaky-SCID or variant
SCID. The test is not indicated for screening B-cell
deficiency disorders other than XLA, such as
atypical XLA, or for screening of XLA carriers.
This test is not intended for use as a diagnostic test
and a positive screening result should be followed
by confirmatory testing. | The EnLite™ Neonatal TREC Kit is an
in vitro diagnostic device intended
for the semi-quantitative
determination of TREC (T-cell
receptor excision circle) DNA in blood
specimens dried on filter paper. The
test is for use on the VICTOR™ EnLite
instrument. The test is indicated for
use as an aid in screening newborns
for severe combined
immunodeficiency disorder (SCID).
This test is not intended for use as a
diagnostic test or for screening of
SCID-like Syndromes, such as
DiGeorge Syndrome, or Omenn
Syndrome. It is also not intended to
screen for less acute SCID syndromes
such as leaky-SCID or variant SCID. |
| Test
Methodology | Semi-quantitative, multiplex real-time fluorescent-
based polymerase chain reaction (PCR) based
nucleic acid amplification and detection | Semi-quantitative, polymerase chain
reaction (PCR) based nucleic acid
amplification and time-resolved
fluorescence resonance energy
transfer (TR-FRET) based detection |
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| Instrument /
Software
Platform | QuantStudio™ Dx Real-Time PCR instrument
(K123955) and Eonis Analysis Software | VICTOR™ EnLite instrument and the
EnLite™ workstation software. | | | | | | | | | | | | |
|--------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------|--------|-----|-------|------------------------|------|------|-----------------------------------------|------|--------|------|------------------------|------|
| Sample Type | Punch from dried blood spot (DBS) specimen | Same | | | | | | | | | | | | |
| Reportable
Range | TREC 242 – 4320 copies/105 cells
KREC 459 - 24300 copies/105 cells | TREC 29-473 copies/µL blood | | | | | | | | | | | | |
| Lower Limits
of Measure | TREC LoB=0 copies/105 cells, LoD=LoQ=242
copies/105 cells
KREC LoB=0 copies/105 cells, LoD=LoQ=459
copies/105 cells | TREC LoB=3 copies/ µL blood,
LoD=20 copies/ µL blood, LoQ=29
copies/ µL blood | | | | | | | | | | | | |
| Calibrators /
Standards | Calibration is based on internal reference (RPP30)
in each well and manufacturer calibration for each
kit lot. | 3 levels of DBS calibrators prepared
from porcine whole blood spiked
with TREC and beta-actin (reference)
plasmids, and a no-template blank | | | | | | | | | | | | |
| Controls | 3 levels of DBS controls prepared from leucocyte-
depleted human red blood cells and TREC, KREC,
SMN1 and RPP30 plasmids spiked in, plus No
template control (NTC) | 3 levels of DBS controls prepared
from porcine whole blood, TREC and
beta-actin (reference) plasmids
spiked in. | | | | | | | | | | | | |
| Expected
Values | Analyte
copies/105
cells | N | Median | Min | Max | 0.3% | 0.5% | 1.0% | Analyte
copies/µL
blood | N | Median | 2.0% | 2.5% | 5.0% |
| | TREC | 3341 | 2520 | 117 | 9990 | 262 | 413 | 563 | TREC | 2846 | 150 | 34 | 36 | 46 |
| | KREC | 3341 | 3590 | 12 | 20100 | 129 | 261 | 484 | | | | | | |
| Reproducibility | | | | | | | | | | | | | | |
| | | Sample
Range (copies/105
cells) | | | | Lognormal %CV
Range | | | Sample
Range
(copies/µL
blood) | | | | Lognormal
%CV Range | |
| | TREC | 299 - 3867 | | | | 27%-65% | | | TREC 56 - 545 | | | | 49%-87% | |
| | KREC | 763 - 9648 | | | | 26%-50% | | | | | | | | |
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4. Summary of the Studies
SITE-TO-SITE REPRODUCIBILITY
The reproducibility of the Eonis SCID-SMA assay was determined using a panel of dried blood spots at different TREC, KREC levels, 1 reagent kit lot, 2 external newborn screening laboratories and 1 internal site. In each laboratory, 2 operators performed 5 runs each during 5 operating days. Each run consisted of 1 plate with 5 replicates per sample. Total number of measurements was 150 per sample in each laboratory). The analysis of variance approach was used to calculate the following:
The values in the mean TREC and KREC copies/10° cells column are transformed from logarithmic (Ln) mean values, and therefore they represent geometric means in the copies/105 cells scale. The analysis of variance approach was used to calculate the results presented as SDs in the logarithmic (Ln) scale complemented with total %CV in lognormal scale. Summary mean, min and max copies/105 cells and %CV results without log transformation for total imprecision are also shown.
| Calculations with logarithmic transformation | ||||||||
|---|---|---|---|---|---|---|---|---|
| Sample | Geometric | |||||||
| Mean | ||||||||
| (Copies | ||||||||
| 105/cells) | Mean Ln | |||||||
| (Copies | ||||||||
| 105/cells) | Repeatability Ln SD | Between | ||||||
| Run Ln SD | Between | |||||||
| Operator | ||||||||
| Ln SD | Between | |||||||
| Site Ln SD | Total | |||||||
| Ln SD | Log- | |||||||
| normal | ||||||||
| CV% | ||||||||
| 6 | 23 | 3.14 | 0.73 | 0.60 | 0.01 | 0.53 | 1.09 | 151 |
| 13 | 34 | 3.53 | 1.12 | 0.31 | 0.00 | 0.32 | 1.20 | 180 |
| 10 | 180 | 5.19 | 0.77 | 0.01 | 0.00 | 0.03 | 0.77 | 90 |
| 5 | 299 | 5.70 | 0.55 | 0.16 | 0.01 | 0.15 | 0.59 | 65 |
| 3 | 912 | 6.82 | 0.29 | 0.06 | 0.00 | 0.10 | 0.31 | 32 |
| 12 | 936 | 6.84 | 0.34 | 0.00 | 0.00 | 0.18 | 0.38 | 40 |
| 4 | 1026 | 6.93 | 0.28 | 0.10 | 0.01 | 0.13 | 0.33 | 34 |
| 2 | 1478 | 7.30 | 0.26 | 0.01 | 0.04 | 0.04 | 0.27 | 27 |
| 8 | 3013 | 8.01 | 0.48 | 0.20 | 0.01 | 0.16 | 0.55 | 59 |
| 11 | 3290 | 8.10 | 0.25 | 0.05 | 0.00 | 0.09 | 0.27 | 28 |
| 7 | 3867 | 8.26 | 0.29 | 0.08 | 0.00 | 0.11 | 0.32 | 33 |
| 9 | 8225 | 9.01 | 0.24 | 0.08 | 0.02 | 0.18 | 0.31 | 32 |
| 1 | 9000 | 9.11 | 0.21 | 0.08 | 0.00 | 0.14 | 0.26 | 27 |
TREC Reproducibility data pooled across three laboratories.
TREC total variation results without logarithmic transformation.
| Calculations without logarithmic transformation | ||||||
|---|---|---|---|---|---|---|
| Sample | N | Mean | ||||
| (Copies/ | ||||||
| 105 cells) | Min (Copies/ | |||||
| 105 cells) | Max (Copies/ | |||||
| 105 cells) | SD | |||||
| (Copies/ | ||||||
| 105 cells) | CV% | |||||
| 6 | 54 | 40 | 4 | 308 | 55.3 | 138 |
| 13 | 71 | 58 | 3 | 239 | 56 | 97 |
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Image /page/9/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue stylized "P" shape that resembles a forward-pointing arrow or play button. Below the symbol, the word "PerkinElmer" is written in a bold, sans-serif font. Underneath "PerkinElmer", the tagline "For the Better" is written in a smaller, italicized font.
| Calculations without logarithmic transformation | ||||||
|---|---|---|---|---|---|---|
| Sample | N | Mean | ||||
| (Copies/ | ||||||
| 105 cells) | Min (Copies/ | |||||
| 105 cells) | Max (Copies/ | |||||
| 105 cells) | SD | |||||
| (Copies/ | ||||||
| 105 cells) | CV% | |||||
| 10 | 148 | 223 | 5 | 796 | 126 | 56 |
| 5 | 150 | 346 | 41 | 971 | 174 | 50 |
| 3 | 150 | 952 | 220 | 1792 | 272 | 29 |
| 12 | 150 | 997 | 239 | 2563 | 349 | 35 |
| 4 | 150 | 1076 | 497 | 2490 | 335 | 31 |
| 2 | 150 | 1526 | 654 | 2411 | 370 | 24 |
| 11 | 150 | 3388 | 513 | 6401 | 767 | 23 |
| 7 | 150 | 4076 | 2043 | 16934 | 1590 | 39 |
| 8 | 150 | 4601 | 797 | 218936 | 17700 | 384* |
| 9 | 150 | 8571 | 3233 | 14974 | 2420 | 28 |
| 1 | 150 | 9277 | 4236 | 16270 | 2320 | 25 |
*Dataset for sample 8 has one high outlier (max value) affecting the variability estimate. Without the outlier, the estimated CV is 41% and similar to other samples within the measuring range.
| KREC Reproducibility data pooled across three laboratories. | ||
|---|---|---|
| ------------------------------------------------------------- | -- | -- |
| Calculations with logarithmic transformation | ||||||||
|---|---|---|---|---|---|---|---|---|
| Sample | Geometric | |||||||
| Mean (Copies | ||||||||
| 105/cells) | Mean Ln | |||||||
| (Copies | ||||||||
| 105/cells) | Repeata- | |||||||
| bility | ||||||||
| Ln SD | Between | |||||||
| Run | ||||||||
| Ln SD | Between | |||||||
| Operator | ||||||||
| Ln SD | Between | |||||||
| Site | ||||||||
| Ln SD | Total Ln | |||||||
| SD | Log- | |||||||
| normal | ||||||||
| CV% | ||||||||
| 10 | 348 | 5.85 | 0.72 | 0.01 | 0.13 | 0.22 | 0.76 | 89 |
| 8 | 763 | 6.64 | 0.46 | 0.05 | 0.00 | 0.13 | 0.48 | 50 |
| 13 | 792 | 6.67 | 0.43 | 0.09 | 0.06 | 0.09 | 0.45 | 47 |
| 12 | 2770 | 7.93 | 0.24 | 0.00 | 0.00 | 0.17 | 0.30 | 31 |
| 3 | 3849 | 8.26 | 0.28 | 0.07 | 0.00 | 0.09 | 0.31 | 31 |
| 7 | 4037 | 8.30 | 0.23 | 0.02 | 0.05 | 0.11 | 0.26 | 27 |
| 1 | 4359 | 8.38 | 0.19 | 0.08 | 0.03 | 0.06 | 0.22 | 22 |
| 6 | 5007 | 8.52 | 0.38 | 0.01 | 0.00 | 0.06 | 0.38 | 40 |
| 4 | 9313 | 9.14 | 0.20 | 0.05 | 0.00 | 0.02 | 0.21 | 21 |
| 11 | 9648 | 9.17 | 0.25 | 0.05 | 0.00 | 0.05 | 0.26 | 26 |
| 5 | 16531 | 9.71 | 0.19 | 0.07 | 0.06 | 0.13 | 0.24 | 25 |
| 2 | 34982 | 10.5 | 0.27 | 0.00 | 0.01 | 0.01 | 0.27 | 28 |
| 10 | 348 | 5.85 | 0.72 | 0.01 | 0.13 | 0.22 | 0.76 | 89 |
KREC total variation results without logarithmic transformation.
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Image /page/10/Picture/1 description: The image shows the logo for PerkinElmer. The logo consists of a blue abstract shape above the company name, "PerkinElmer," which is written in a bold, sans-serif font. Below the company name is the tagline "For the Better" in a smaller, italicized, blue font. The abstract shape above the name is a blue triangle with a curved shape on the right side.
| Sample | N | Mean
(Copies/
105 cells) | Min (Copies/
105 cells) | Max (Copies/
105 cells) | SD
(Copies/
105 cells) | CV% |
|--------|-----|--------------------------------|----------------------------|----------------------------|------------------------------|-----|
| 10 | 148 | 431 | 40 | 1071 | 242 | 56 |
| 8 | 150 | 841 | 186 | 1925 | 349 | 41 |
| 13 | 150 | 862 | 183 | 2003 | 330 | 38 |
| 12 | 150 | 2877 | 1097 | 5232 | 792 | 28 |
| 3 | 150 | 4025 | 1075 | 13447 | 1300 | 32 |
| 7 | 150 | 4162 | 2241 | 6788 | 1030 | 25 |
| 1 | 150 | 4457 | 2104 | 7746 | 960 | 22 |
| 6 | 150 | 5457 | 1567 | 36630 | 3230 | 59 |
| 4 | 150 | 9508 | 4735 | 15856 | 1910 | 20 |
| 11 | 150 | 9915 | 1564 | 16967 | 2130 | 21 |
| 5 | 150 | 16955 | 8813 | 29034 | 3880 | 23 |
| 2 | 150 | 36370 | 16841 | 118413 | 11200 | 31 |
PRECISION
The quantitative precision was determined in accordance with CLSI document EP05-A3.
The variation of the Eonis SCID-SMA assay was determined using dried blood spot samples, 3 kit lots, 3 sets of Eonis test systems (including three JANUS Extraction Instruments, three JANUS PCR Mastermix Instruments and three QuantStudio™ Dx Real-Time PCR Instruments), 2 operators, and 54 runs over 23 calendar days. Each run consisted of 1 plate with 2 replicates per sample in a randomized plate map. Total number of measurements was 108 per sample. The analysis of variance approach was used to calculate the following:
The values in the mean TREC and KREC copies/10° cells column are transformed from logarithmic (Ln) mean values, and therefore they represent geometric means in the copies/105 cells scale. The analysis of variance approach was used to calculate the results presented as SDs in the logarithmic (Ln) scale complemented with total %CVs in lognormal scale. Summary mean, min and max copies/10 cells and SD and %CV results without log transformation for total imprecision are also shown.
| Calculations with logarithmic transformation | ||||||||
|---|---|---|---|---|---|---|---|---|
| Sample | Geometric | |||||||
| Mean (Copies | ||||||||
| 105/cells) | Mean Ln | |||||||
| (Copies | ||||||||
| 105/cells) | Repeatability | |||||||
| Ln SD | Between | |||||||
| Run | ||||||||
| Ln SD | Between | |||||||
| Instrument | ||||||||
| Ln SD | Between | |||||||
| Lot | ||||||||
| Ln SD | Total Ln | |||||||
| SD | Log- | |||||||
| normal | ||||||||
| CV% | ||||||||
| 6 | 20 | 3.00 | 0.92 | 0.00 | 0.00 | 0.33 | 0.97 | 125 |
| 13 | 34 | 3.54 | 0.89 | 0.00 | 0.00 | 0.23 | 0.92 | 116 |
| 10 | 159 | 5.07 | 0.76 | 0.17 | 0.00 | 0.14 | 0.79 | 92 |
| 5 | 464 | 6.14 | 0.42 | 0.06 | 0.14 | 0.13 | 0.47 | 49 |
TREC Precision data across three kit lots and three instruments.
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| 12 | 1022 | 6.93 | 0.34 | 0.16 | 0.00 | 0.08 | 0.39 | 40 |
|---|---|---|---|---|---|---|---|---|
| 2 | 1130 | 7.03 | 0.35 | 0.20 | 0.00 | 0.02 | 0.40 | 42 |
| 4 | 1176 | 7.07 | 0.33 | 0.13 | 0.13 | 0.15 | 0.41 | 42 |
| 3 | 2165 | 7.68 | 0.37 | 0.00 | 0.00 | 0.05 | 0.37 | 38 |
| 11 | 4105 | 8.32 | 0.24 | 0.08 | 0.16 | 0.04 | 0.30 | 30 |
| 7 | 4146 | 8.33 | 0.32 | 0.15 | 0.10 | 0.01 | 0.37 | 38 |
| 8 | 4866 | 8.49 | 0.35 | 0.31 | 0.00 | 0.07 | 0.47 | 50 |
| 9 | 8604 | 9.06 | 0.30 | 0.35 | 0.19 | 0.18 | 0.54 | 58 |
| 1 | 11048 | 9.31 | 0.21 | 0.14 | 0.16 | 0.03 | 0.30 | 31 |
TREC total variation results without logarithmic transformation.
| Sample | N | Mean (Copies/ 105 cells) | Min (Copies/ 105 cells) | Max (Copies/ 105 cells) | SD (Copies/ 105 cells) | CV% |
|---|---|---|---|---|---|---|
| 6 | 29 | 32 | 5 | 150 | 34.9 | 109 |
| 13 | 43 | 49 | 4 | 173 | 41.4 | 84 |
| 10 | 105 | 200 | 11 | 479 | 114 | 57 |
| 5 | 107 | 508 | 99 | 1183 | 216 | 43 |
| 12 | 106 | 1094 | 189 | 2247 | 383 | 35 |
| 2 | 107 | 1206 | 230 | 2576 | 414 | 34 |
| 4 | 108 | 1259 | 422 | 3011 | 479 | 38 |
| 3 | 107 | 2263 | 107 | 3084 | 510 | 23 |
| 11 | 106 | 4263 | 2031 | 7418 | 1220 | 29 |
| 7 | 107 | 4407 | 1014 | 8206 | 1530 | 35 |
| 8 | 107 | 5754 | 2011 | 74368 | 7120 | 124 |
| 9 | 107 | 9540 | 471 | 19530 | 3760 | 39 |
| 1 | 107 | 11501 | 3218 | 19982 | 3170 | 28 |
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Image /page/12/Picture/1 description: The image shows the logo for PerkinElmer. The logo features a stylized blue "P" shape, with a vertical bar and an arrow-like shape pointing to the right. Below the symbol is the company name "PerkinElmer" in a bold, sans-serif font, with the "E" in "Elmer" being in blue. Underneath the company name is the tagline "For the Better" in a smaller, italicized font, also in blue.
| Calculations with logarithmic transformation | ||||||||
|---|---|---|---|---|---|---|---|---|
| Sample | Geometric | |||||||
| Mean | ||||||||
| (Copies | ||||||||
| 105/cells) | Mean Ln | |||||||
| (Copies | ||||||||
| 105/cells) | Repeata- | |||||||
| bility | ||||||||
| Ln SD | Between | |||||||
| Run | ||||||||
| Ln SD | Between | |||||||
| Instrument | ||||||||
| SD | Between | |||||||
| Lot | ||||||||
| Ln SD | Total Ln | |||||||
| SD | Log- | |||||||
| normal | ||||||||
| CV% | ||||||||
| 9 | 85 | 4.44 | 1.14 | 0.00 | 0.00 | 0.37 | 1.20 | 178 |
| 10 | 478 | 6.17 | 0.53 | 0.36 | 0.00 | 0.16 | 0.66 | 74 |
| 13 | 1033 | 6.94 | 0.40 | 0.13 | 0.15 | 0.09 | 0.45 | 48 |
| 8 | 1075 | 6.98 | 0.47 | 0.00 | 0.00 | 0.13 | 0.49 | 52 |
| 7 | 2416 | 7.79 | 0.43 | 0.00 | 0.08 | 0.01 | 0.44 | 46 |
| 12 | 3361 | 8.12 | 0.36 | 0.10 | 0.00 | 0.07 | 0.38 | 39 |
| 1 | 5271 | 8.57 | 0.35 | 0.00 | 0.09 | 0.04 | 0.37 | 38 |
| 3 | 5541 | 8.62 | 0.30 | 0.05 | 0.00 | 0.03 | 0.31 | 31 |
| 6 | 8691 | 9.07 | 0.26 | 0.26 | 0.08 | 0.10 | 0.39 | 40 |
| 4 | 11499 | 9.35 | 0.29 | 0.18 | 0.12 | 0.06 | 0.36 | 38 |
| 11 | 13494 | 9.51 | 0.24 | 0.08 | 0.17 | 0.09 | 0.32 | 32 |
| 5 | 13767 | 9.53 | 0.22 | 0.21 | 0.13 | 0.07 | 0.34 | 35 |
| 2 | 39735 | 10.6 | 0.20 | 0.21 | 0.12 | 0.07 | 0.32 | 33 |
KREC Precision data across three kit lots and three instruments.
KREC total variation results without logarithmic transformation.
| Calculations without logarithmic transformation | ||||||
|---|---|---|---|---|---|---|
| Sample | N | Mean | ||||
| (Copies/ | ||||||
| 105 cells) | Min (Copies/ | |||||
| 105 cells) | Max (Copies/ | |||||
| 105 cells) | SD | |||||
| (Copies/ | ||||||
| 105 cells) | CV% | |||||
| 9 | 10 | 146 | 17 | 435 | 139 | 95 |
| 10 | 104 | 570 | 43 | 1396 | 306 | 54 |
| 13 | 106 | 1139 | 212 | 4188 | 546 | 48 |
| 8 | 107 | 1195 | 356 | 3180 | 538 | 45 |
| 7 | 107 | 2605 | 203 | 5513 | 898 | 34 |
| 12 | 107 | 3568 | 686 | 7096 | 1150 | 32 |
| 1 | 107 | 5560 | 474 | 10461 | 1620 | 29 |
| 3 | 106 | 5774 | 863 | 10031 | 1500 | 26 |
| 6 | 107 | 9308 | 3333 | 19079 | 3420 | 37 |
| 4 | 108 | 12212 | 2707 | 23846 | 4180 | 34 |
| 11 | 107 | 14103 | 5330 | 23372 | 4030 | 29 |
| 5 | 106 | 14492 | 5673 | 35070 | 4960 | 34 |
| 2 | 108 | 41717 | 15231 | 74089 | 12400 | 30 |
Qualitative imprecision was determined in accordance with CLSI document EP12-A2 to classify the results, using the screening performance study cut-offs (262 copies/105 cells for TREC, 484 copies/108 cells for KREC). The C5-C95 interval was determined to be 79–626 copies/105 cells for TREC, and 189–1064 copies/105 cells for KREC.
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Concentrations outside of these intervals were consistently analyte negative (concentrations C95, equals to screen negative).
LIMIT OF DETECTION
The Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) were determined in accordance with CLSI document EP17-A2. The data for LoB was analyzed using "Assign LoB = Zero and Confirm" approach. The data for LoD was analyzed with probit approach.
Based on 300 determinations of blank samples (150 for each kit lot) the limit of blank (LoB) for TREC, KREC, SMN1 is 0 copies/μL blood and 0 copies/105 cells.
Based on total of 960 determinations, 20 replicates per dilution, the limit of detection (LoD) for TREC is 242 copies/10° cells with 95% probability. The Limit of Quantitation (LoQ) for TREC is 242 copies/10° cells, which is equal to LoD. The limit of detection (LoD) for KREC is 459 copies/105 cells) with 95% probability. The Limit of Quantitation (LoQ) for KREC is 459 copies/105 cells, which is equal to LoD.
LINEARITY
Linearity was determined in accordance with CLSI document EP06 ED2:2020 with one kit lot. Three (3) sets of contrived samples were used for this evaluation. Two sample sets were diluted to 9 levels and tested with 4 replicates for each level. For KREC the linearity panel was amended with results from LoD-study to reach lower concentrations. The allowable maximum deviation from linearity in the study was 25%.
The Eonis SCID-SMA assay is demonstrated to be linear for TREC from 94 copies/105 cells with observed maximum deviation of -14.3%. The KREC analyte is demonstrated to be linear from 117 to 24343 copies/10° cells with observed maximum deviation of 17.3%.
INTERFERENCE
The Eonis SCID-SMA kit was evaluated for interference from potential endogenous and exogenous sources in accordance with CLSI document EP07-A3.
The following potentially interfering substances were added to whole blood spiked with at three different TREC plasmid and KREC plasmid concentrations and were found not to interfere at the concentration indicated.
Interference substances tested and their concentrations
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| Tested substance | Added concentration of tested substance |
|---|---|
| Conjugated bilirubin | 16.6 mg/dL in blood |
| Hemoglobin | 200 g/L in blood |
| Unconjugated bilirubin | 10 mg/dL in blood |
| Intralipid® | 1500 mg/dL in blood |
| Li-heparin | 7500 USP /dL in blood |
| EDTA | 9.8 mg/mL in blood |
| Na-citrate | 0.0645 mol/L in blood |
SCREENING PERFORMANCE
The screening performance of the Eonis SCID-SMA kit was determined in a clinical study conducted in Denmark. Retrospective archived dried blood spot specimens (collected from US and Denmark) from subjects confirmed positive for SCID, XLA were included to enrich the cohort of routine newborn screening specimens obtained from the Danish Newborn Screening Biobank.
Confirmatory test results were used as the comparator for the confirmed positive SCID, XLA cases. The clinical status of the routine subjects was determined through a retrospective review by clinical experts to confirm the routine subject cohort samples were from unaffected individuals.
Using the data collected to establish the expected values of the Eonis SCID-SMA kit were determined by calculating the TREC concentrations corresponding to the 0.3" and 1.0" population percentiles (262 copies/10 cells for TREC, 484 copies/10 cells for KREC) established in a cut-off study with an independent dataset. The specimens having TREC and KREC levels below the cut-off values in the initial round of testing were re-tested in duplicate. The final results (presumptive positive positive, invalid result) were classified after the second round of testing. Summary of the retest rate and final results for routine screening specimens is provided below.
Intralipid is a registered trademark of Fresenius Kabi AB.
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| Routine screening samples | SMN1 | TREC | KREC |
|---|---|---|---|
| Screened samples | 3018 | 3018 | 3018 |
| Initial screen positive | 0 | 10 | 18 |
| Initial screen negative | 3018 | 3008 | 3000 |
| Retest rate | 0% | 0.3% | 0.6% |
| Final screen positive | 0 | 9 | 9 |
| Final screen negative | 3018 | 3008 | 3004 |
| False-positive rate | 0% | 0.3% | 0.3% |
Summary of the samples tested in the pivotal study.
The screening performance of the Eonis SCID-SMA kit was established by measuring TREC, KREC (and RPP30) in 3090 DBS specimens.
In total, 17 SCID and 6 XLA retrospective case specimens and 3018 normal newborn screening specimens were available for the study. The screening performance of the Eonis SCID-SMA kit was established by measuring TREC, and KREC (and RPP30) and the final results after retesting are presented below.
Screening performance of Eonis SCID-SMA kit.
| TREC | Clinical Status | Total (%) | ||
|---|---|---|---|---|
| SCID affected (%) | Normal (%) | |||
| Screening | ||||
| result | Presumptive positive (%) | 17 (100 %) | 9 (0.3 %) | 26 (0.9 %) |
| Presumptive normal (%) | 0 (0.0 %) | 3008 (99.7 %) | 3008 (99.1 %) | |
| Total (%) | 17 (100 %) | 3017 (100 %) | 3034 (100 %) |
| KREC | Clinical Status | |||
|---|---|---|---|---|
| XLA affected (%) | Normal (%) | Total (%) | ||
| Screening | ||||
| result | Presumptive positive (%) | 6 (100 %) | 9* (0.3 %) | 15 (0.5 %) |
| Presumptive normal (%) | 0 (0.0 %) | 3004 (99.7 %) | 3004 (99.5 %) | |
| Total (%) | 6 (100 %) | 3013 (100 %) | 3019 (100 %) |
*Includes one false positive result from a female population the false positive rate is estimated to be 0.1 %
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| KREC
| (Male only) | Clinical Status | |||
|---|---|---|---|---|
| XLA affected (%) | Normal (%) | Total (%) | ||
| Screening | ||||
| result | Presumptive positive (%) | 6 (100 %) | 8 (0.5 %) | 14 (0.9 %) |
| Presumptive normal (%) | 0 (0.0 %) | 1515 (99.5 %) | 1515 (99.1 %) | |
| Total (%) | 6 (100 %) | 1523 (100 %) | 1529 (100 %) |
Conclusion
The Eonis SCID-SMA kit demonstrates analytical and screening performance that supports its substantial equivalency with the predicate device, PerkinElmer ENLITE™ Neonatal TREC Kit (DEN140010).