Eonis SCID-SMA kit

K203035 · Perkinelmer, Inc. · PJI · Nov 9, 2022 · Immunology

Device Facts

• Record ID: K203035
• Device Name: Eonis SCID-SMA kit
• Applicant: Perkinelmer, Inc.
• Product Code: PJI · Immunology
• Decision Date: Nov 9, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5930
• Device Class: Class 2
• Attributes: Pediatric

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.

Device Story

The Eonis SCID-SMA kit is an in vitro diagnostic assay for newborn screening. It processes 3.2 mm punches from dried blood spots (DBS) on filter paper. DNA is extracted using the Eonis DNA Extraction kit, either manually or via the JANUS G3 Automated Workstation. The assay uses multiplex real-time PCR with target-specific primers and TaqMan probes to detect TREC, KREC, and the RPP30 internal control gene. The QuantStudio Dx Real-Time PCR instrument measures fluorescence intensity, which the Eonis Analysis Software converts into cycle threshold (Ct) values. The software calculates TREC and KREC concentrations (copies/10⁵ cells) relative to RPP30. Results are interpreted against predefined cut-offs to identify presumptive positive or normal samples. Presumptive positives require confirmatory testing. The device aids clinicians in early identification of SCID and XLA, facilitating timely intervention.

Clinical Evidence

Clinical study of 3090 DBS specimens (17 SCID, 6 XLA, 3018 normal). TREC sensitivity 100% (CI 80.5%-NA), specificity 99.7% (CI 99.4%-99.9%). KREC sensitivity 100% (CI 54.1%-NA), specificity 99.7% (CI 99.4%-99.9%). Performance established via retrospective review of clinical status.

Technological Characteristics

Multiplex real-time PCR assay; TaqMan probe-based detection; QuantStudio Dx Real-Time PCR instrument; automated DNA extraction (JANUS G3); internal control (RPP30) for normalization; results reported in copies/10⁵ cells; storage at -30°C to -16°C.

Indications for Use

Indicated for newborn screening for Severe Combined Immunodeficiency (SCID) via TREC determination and X-linked agammaglobulinemia (XLA) via KREC determination using DNA from dried blood spots. Not for screening SCID-like syndromes (e.g., DiGeorge, Omenn), leaky/variant SCID, non-XLA B-cell deficiencies, or XLA carriers.

Regulatory Classification

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.

Special Controls

*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.

Predicate Devices

• PerkinElmer EnLite™ Neonatal TREC Kit (DEN140010)

Submission Summary (Full Text)

{0}

FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number

K203035

B Applicant

PerkinElmer Inc

C Proprietary and Established Names

Eonis SCID-SMA kit

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PJI | Class II | 21 CFR 866.5980 - Newborn Screening Test for Severe Combined Immunodeficiency Disorder (SCID) | IM - Immunology |

## II Submission/Device Overview:

A Purpose for Submission:

New Device

B Measurand:

T-cell receptor excision circles (TRECs) and Kappa-deleting element recombination circles (KRECs)

C Type of Test:

Multiplex real-time polymerase chain reaction-based assay using target sequence specific primers and probes

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

{1}

K203035 - Page 2 of 43

## III Intended Use/Indications for Use:

### A Intended Use(s):

See Indications for Use below.

### B Indication(s) 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.

### C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

In vitro diagnostic use

### D Special Instrument Requirements:

QuantStudio™ Dx Real-Time PCR Instrument (ThermoFisher, k123955)

Liquid Handling Instruments: JANUS G3 Automated Workstation Extraction instrument (labeled for IVD use) or JANUS Extraction Mini instrument (labeled for IVD use) and the JANUS PCR Mastermix instrument (labeled for IVD use).

## IV Device/System Characteristics:

### A Device Description:

The Eonis SCID-SMA kit contains reagents to detect three biomarkers: TREC, KREC and exon 7 in the SMNI gene. Detection of SMN1 gene was submitted and reviewed in DEN200044. This Decision Summary only discusses the validation data in support of TREC and KREC.

The newborn screening workflow for the Eonis SCID-SMA kit includes:

- Two liquid handling platforms (one for DNA extraction and one for PCR master mix setup)
- QuantStudio™ Dx Real-Time PCR instrument (k123955)
- Eonis Analysis Software

{2}

Each Eonis SCID-SMA kit contains reagents for up to 384 reactions or 1152 reactions including kit controls. The kit contents are listed in Table 1. Materials required but not provided include the Eonis DNA Extraction Kit, Eonis Analysis Software, and consumables.

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/SMN1) control | |
| C2 Low TREC/KREC and normal SMN1 control | |
| C3 High TREC/KREC and normal SMN1 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 |

B Principle of Operation:

The Eonis SCID-SMA kit is a multiplex real-time PCR based assay that uses target sequence-specific primers and TaqMan™ probes to amplify and detect TREC, KREC, and RPP30 in DNA extracted from newborn dried blood spot (DBS) using the Eonis DNA Extraction kit in a single PCR reaction. Each TaqMan probe has a unique dye linked to their terminal 5′ end, allowing for the simultaneous detection of the four targets, if present. The amount of each target present in the DNA is determined by the intensity of fluorescence emitted by each dye released from the degraded probe during amplification and detected by the QuantStudio Dx Real-Time PCR instrument. The instrument measures fluorescence signals and converts them into comparative quantitative readouts which are expressed as a function of cycle threshold (Ct) values. RPP30 is used as an internal amplification control for DNA quality and to relatively quantitate TREC and KREC (copies/10⁵ cells) using TREC ΔCt and KREC ΔCt. TREC and KREC concentrations are calculated using the following formulas to generate results expressed as copies/10⁵ cells:

- TREC: 2 X 2⁻(ΔCt TREC) x 117000
- KREC: 2 X 2⁻(ΔCt TREC) x 254000

The interpretation of results based on copy number is shown in Tables 2 and 3.

K203035 - Page 3 of 43

{3}

Table 2. TREC result interpretation matrix

| RPP30 Ct values | | TREC Ct values | | TREC conc. (copies/105cells) | Result interpretation for TREC |
| --- | --- | --- | --- | --- | --- |
| Ct < 15.0 | Values not considered when RPP30 Ct < 15.0 | | | | Invalid* |
| 15.0 ≤ Ct ≤ 32.00 | AND | Ct < 18.0 | Values not considered when TREC Ct < 18.0 | | Invalid* |
| | AND | Ct ≥ 18.0 | AND | TREC conc. ≥ CutoffTREC | Presumptive normal |
| | | | AND | TREC conc. < CutoffTREC | Presumptive positive* |
| | AND | No Ct | N/A | | Presumptive positive* |
| Ct > 32.00 or no Ct | Values not considered when RPP30 Ct > 32.00 or no Ct | | | | Invalid* |

*Samples with invalid results should be repeated in singlicate, and samples with presumptive positive results should be repeat tested in duplicate. If one of the duplicate samples is positive, the result is presumptive positive.

Table 3. KREC result interpretation matrix

| RPP30 Ct values | | KREC Ct values | | KREC conc. (copies/105cells) | Result interpretation for KREC |
| --- | --- | --- | --- | --- | --- |
| Ct < 15.0 | Values not considered when RPP30 Ct < 15.0 | | | | Invalid* |
| 15.0 ≤ Ct ≤ 32.00 | AND | Ct < 18.0 | Values not considered when TREC Ct < 18.0 | | Invalid* |
| | AND | Ct ≥ 18.0 | AND | KREC conc. ≥ CutoffKREC | Presumptive normal |
| | | | AND | KREC conc. < CutoffKREC | Presumptive positive* |
| | AND | No Ct | N/A | | Presumptive positive* |
| Ct > 32.00 or no Ct | Values not considered when RPP30 Ct > 32.00 or no Ct | | | | Invalid* |

*Samples with invalid results should be repeated in singlicate, and samples with presumptive positive results should be repeat tested in duplicate. If one of the duplicate samples is positive, the result is presumptive positive.

K203035 - Page 4 of 43

{4}

K203035 - Page 5 of 43

## Result Reporting

Samples that result in values below 242 copies/10⁵ cells are recommended to be reported as "&lt;242 copies/10⁵ cells". Samples that result in values above 4316 copies/10⁵ cells are recommended to be reported as "&gt;4320 copies/10⁵ cells" and are considered to be TREC screen negative.

Samples that result in values below 459 copies/10⁵ cells are recommended to be reported as "&lt;459 copies/10⁵ cells". Samples that result in values above 24343 copies/10⁵ cells are recommended to be reported as "&gt;24300 copies/10⁵ cells" and are considered to be KREC screen negative.

## C Instrument Description Information:

1. **Instrument Name:**

QuantStudio Dx Real-Time PCR instrument (The information below is specific to the TREC and KREC analytes; refer to the decision summary for additional information regarding the features in this section K123955)

JANUS G3 Automated Workstation (for IVD use) consisting of the JANUS Extraction Instrument and JANUS PCR Mastermix Instrument

2. **Specimen Identification:**

Specimens are barcoded and entered into the testing queue consistent with the laboratory procedures.

3. **Specimen Sampling and Handling:**

The required sample is a 3.2 mm specimen punch from a dried blood spot (DBS) into a 96-well plate. DNA is extracted and purified using the Eonis DNA Extraction kit. The extraction can be completed manually or using an automated liquid handling platform, the JANUS G3 Workstation (IVD), for processing up to four 96-well plates simultaneously.

4. **Calibration:**

The device (Eonis SCID-SMA kit) calibrates the TREC results utilizing the internal control RPP30 gene (assumes two copies) and a mathematical algorithm and reports out the results in the unit of copies/10⁵ cells.

5. **Quality Control:**

Quality control is addressed for each separately cleared specific assay to be run on the instrument. For the Eonis SCID-SMA assay, quality is maintained by the process control, used during sample processing and amplification in the assay. Additionally, there are external positive and negative controls available that are used in accordance with the assay instructions for use.

{5}

Controls are used throughout the entire procedure from sample preparation to PCR as a quality check for performance of the Eonis test. The Eonis SCID-SMA kit contains three multi-level dried blood spot controls: C1, C2 and C3. The DBS controls are filter paper cassettes with each control level spotted in duplicate.

- C1 is an analyte-negative control and does not contain TREC, KRECanalytes.
- C2 and C3 are analyte-positive controls that have different levels of TREC and KREC analytes (lower level in C2 and a higher level in C3).
- All three kit controls have the same level of RPP30 analyte.
- Blank reaction wells without sample disks (NTC [no template control]) are used to monitor the absence of contamination. All controls are prepared from human whole blood with a hematocrit value of 48-55% and contain purified salmon-sperm DNA and synthetic TREC, KREC, and RPP30 plasmid DNA.

Each Eonis run includes all four controls in duplicate in each 96-well DNA extraction plate. The newborn samples and DBS controls are punched into the plate, while the well reserved for the Blank control does not contain any punches. The Blank control undergoes the whole process, from DNA extraction to amplification, and the NTC sample consists of elution solution derived from the DNA extraction process.

The test result validation for each 96-well DNA extraction plate is based on the three kit DBS controls and the Blank reaction well. The Eonis Analysis software will identify each control by their designated locations and check their validity. The possible outcome for each control replicate is 'Valid' or 'Invalid'. If one or more controls are invalid, the results of the clinical specimens processed in the same 96-well DNA extraction plate are reported as 'Invalid'. For the Blank reaction well, both replicates must meet the acceptance criteria for the run to be valid. For C1-C3 DBS controls, the results of both replicates must meet the acceptance criteria for the run to be valid. The thresholds for valid controls are shown in Table 4.

Table 4. Acceptance limits for kit controls for TREC and KREC

| Kit Control Name | Parameter Name | Lower Limit (≥) | Upper Limit (≤ or No Ct) |
| --- | --- | --- | --- |
| NTC (Blank) | TREC Ct | 32.65 | No Ct |
| | KREC Ct | 32.55 | No Ct |
| | RPP30 Ct | 28.35 | No Ct |
| C1 | TREC Ct | 32.65 | No Ct |
| | KREC Ct | 32.55 | No Ct |
| | RPP30 Ct | 15.00 | 28.00 |
| C2 | TREC Ct | 18.00 | No Ct |
| | KREC Ct | 18.00 | No Ct |
| | RPP30 Ct | 15.00 | 28.00 |
| | TREC ΔCt | 6.10 | 13.90 |
| | KREC ΔCt | 3.55 | 16.45 |

K203035 - Page 6 of 43

{6}

K203035 - Page 7 of 43

| Kit Control Name | Parameter Name | Lower Limit (≥) | Upper Limit (≤ or No Ct) |
| --- | --- | --- | --- |
| C3 | TREC Ct | 18.00 | 33.00 |
| | KREC Ct | 18.00 | 34.00 |
| | RPP30 Ct | 15.00 | 28.00 |
| | TREC ΔCt | 1.10 | 8.90 |
| | KREC ΔCt | 0.00 | 12.45 |

V Substantial Equivalence Information:

A Predicate Device Name(s):

PerkinElmer EnLite™ Neonatal TREC Kit

B Predicate 510(k) Number(s):

DEN140010

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K203035 | DEN140010 |
| --- | --- | --- |
| Device Trade Name | Eonis SCID-SMA Kit | EnLite Neonatal TREC Kit |
| General Device Characteristic Similarities | | |
| 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).
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. | 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 for screening of SCID-like Syndromes, such as DiGeorge Syndrome, |

{7}

K203035 - Page 8 of 43
| | It is also not intended to screen for less acute SCID syndromes such as leaky-SCID or variant SCID.
This test is not intended for use as a diagnostic test and a positive screening result should be followed by confirmatory testing. | or Omenn Syndrome. It is also not intended to screen for less acute SCID syndromes such as leaky-SCID or variant SCID. |
| --- | --- | --- |
| **Specimen** | Dried blood spot | Same |
| **Stability/Shelf Life** | The kit should be stored at -30 to -16°C. Once opened, the kit should be used within 14 days. | Same |
| **General Device Characteristic Differences** | | |
| **Intended Use/Indications For Use** | 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 not indicated for screening B-cell deficiency disorders other than XLA, such as atypical XLA, or for screening of XLA carriers. | Not applicable |
| **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 |
| **Units of Reporting** | TREC results are calibrated using the internal control gene and a mathematical algorithm and reports out the results in the unit of copies/10^{5} cells. | TREC results are calibrated against a calibrator curve on each plate and reports out the results in the unit of copies/μL. |
| **Instrument/Software** | QuantStudio Dx Real-Time PCR instrument and Eonis software | VICTOR EnLite instrument and EnLite™ workstation software |
| **Reportable Range** | TREC 242 – 4320 copies/10^{5} cells | TREC 29-473 copies/μL blood |

{8}

| | KREC 459 - 24300 copies/10^{5} cells | |
| --- | --- | --- |
| Lower Limits of Measure | TREC LoB=0 copies/10^{5} cells, LoD/LoQ= 242 copies/10^{5} cells
KREC LoB=0 copies/10^{5} cells, LoD/LoQ =459 copies/10^{5} 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 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. |

VI Standards/Guidance Documents Referenced:

- CLSI EP05-A3: Evaluation of precision of quantitative measurement procedures; approved guideline-third edition
- CLSI EP6-A: Evaluation of the linearity of quantitative measurement procedures: a statistical approach; approved guideline
- CLSI EP07-A2: Interference testing in clinical chemistry: approved guideline-second edition
- CLSI EP25-A: Evaluation of Stability of In Vitro Diagnostic Reagents; approved guideline
- CLSI NBS01-A6: Blood Collection on Filter Paper for Newborn Screening Programs-sixth edition
- CLSI MM13-A Collection Transport Preparation and Storage of Specimens for Molecular Methods; approved guideline
- CLSI MM17 Verification and validation of multiplex nucleic acid assays-second edition
- IEC 62304 Edition 1.1 2015-06 Medial device software-Software life cycle processes

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility:

Study 1: Site-to-Site Reproducibility

The objective of this study was to characterize the within-laboratory precision and reproducibility of the Eonis test system across 3 study sites using a panel of samples with different TREC and KREC levels. At each study site, 2 operators performed 5 runs each, over 5 operating days, 1 run per day, and 5 specimen replicates tested per run. A total of 13

K203035 - Page 9 of 43

{9}

independent samples were tested at each of the 3 study sites using 1 kit lot for a total of 150 replicates per sample (3 sites x 2 operators x 5 runs/days x 5 replicates per run = 150). The study used 1 lot of reagents. The samples were generated to represent a range of concentrations and were spotted onto filter paper, dried overnight, and stored at -30°C to -16°C in a sealed bag with desiccant until use. A description of the samples is provided in Table 5.

Table 5. Descriptive summary samples used in the precision study

| Sample | TREC | KREC | Preparation |
| --- | --- | --- | --- |
| Sample 1 | High | Normal | TREC plasmid spiked into cord blood at 1500 copies/μL with the hematocrit adjusted to 48.2 (approx. Ct value 28.63) |
| Sample 2 | Normal | High | KREC plasmid spiked into cord blood at 5000 copies/μL with the hematocrit adjusted to 48.2 (approx. Ct value 28.11) |
| Sample 3 | Normal | Normal | Cord blood with hematocrit adjusted to 48.2 |
| Sample 4 | Low (Above cut-off) | Normal | Diluting 3mL of cord blood with hematocrit adjusted to 48.2 into 2.25 mL of Sample 6 targeting near but below 98% quantile (32.71) of TREC Ct distribution of a population of presumptive normal NBS samples |
| Sample 5 | Low (Below cut-off) | Normal | Diluting 857.1 μL of cord blood with hematocrit of 48.32 in 2142.9 μL of Sample 6 (approx. Ct value 33.07) |
| Sample 6 | Very low (Near 0) | Normal | KREC plasmid spiked into adult whole blood at 2000 copies/μL with hematocrit adjusted to 50.7 (approx. TREC Ct value 38.08; approx. KREC Ct value 30.56) |
| Sample 7 | Normal | Low (Above cut-off) | Diluting 0.75 mL of cord blood with hematocrit adjusted to 48.2 in 2.25 mL of Sample 8 (approx. KREC Ct value 31.96) |
| Sample 8 | Normal | Low (Below cut-off) | TREC plasmid spiked into adult whole blood at 700 copies/μL with hematocrit adjusted to 50.7 (approx. TREC value 30.01 and approx. KREC value 33.30) |
| Sample 9 | Normal | Very low (Near 0) | TREC plasmid spiked at 2000 copies/μL with hematocrit adjusted to 49.5 (approx. TREC Ct value 30.08 and approx. KREC Ct value 0) |
| Sample 10 | Normal | Normal | Pooled adult whole blood from 2 SMA carriers with hematocrit adjusted to 46.1 |
| Sample 11 | Normal | Normal | TREC plasmid at 700 copies/μL, KREC plasmid at 1000 copies/μL and SMA Coriell cells at 15000 cells/μL spiked into leukocyte depleted blood with hematocrit adjusted to 49.5 |

K203035 - Page 10 of 43

{10}

| Sample | TREC | KREC | Preparation |
| --- | --- | --- | --- |
| Sample 12 | Low (Above cut-off) | Low (Above cut-off) | Diluting 4.4 mL of cord blood with hematocrit adjusted to 48.2 into 6.6 mL of adult whole blood with hematocrit adjusted to 50.7 (approx. TREC Ct value 32.48 and approx. KREC Ct value 31.30) |
| Sample 13 | Low (Below cut-off) | Low (Below cut-off) | Adult blood hematocrit adjusted to 50.7 (approx. TREC Ct value 36.34 and approx. KREC Ct value 33.89) |

The values in the mean TREC and KREC copies/10 $^5$ cells column are transformed from logarithmic (Ln) mean values, and therefore, they represent geometric means in the copies/10 $^5$ cells scale. The analysis of variance approach was used to calculate the results presented as standard deviations (SDs) in the logarithmic (Ln) scale complemented with total coefficient of variation (%CV) in lognormal scale (Table 6 and Table 8). Summary mean, min and max copies/10 $^5$ cells and SD and %CV results without log transformation for total imprecision are shown in Table 7 and Table 9.

# Acceptance Criteria:

- For TREC, the total assay precision within the measuring range must have a $\mathrm{SD} \leq 0.9\mathrm{Ln}$ (copies/10 $^5$ cells) where the measuring range starts at 250 copies/10 $^5$ cells.
- For KREC, the total assay precision within the measuring range must have a $\mathrm{SD} \leq 1.49$ Ln (copies/10 $^5$ cells).

Table 6. TREC reproducibility data pooled across three laboratories

| 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 |

K203035 - Page 11 of 43

{11}

Table 7. TREC total variation results without logarithmic transformation

| | | Calculations without logarithmic transformation | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Sample | N | Mean (Copies/ 105cells) | Min (Copies/ 105cells) | Max (Copies/ 105cells) | SD (Copies/ 105cells) | CV% |
| 6 | 54 | 40 | 4 | 308 | 55.3 | 138 |
| 13 | 71 | 58 | 3 | 239 | 56 | 97 |
| 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.

Table 8. KREC reproducibility data pooled across three laboratories

| 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% |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 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 |

K203035 - Page 12 of 43

{12}

Table 9. KREC total variation results without logarithmic transformation

| | | Calculations without logarithmic transformation | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Sample | N | Mean (Copies/105cells) | Min (Copies/105cells) | Max (Copies/105cells) | SD (Copies/105cells) | 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 |

# Study 2: Lot-to-Lot Reproducibility

A study was performed internally (single site) using 3 sets of Eonis test systems, 2 operators, and 3 kit lots. The 13 samples (Table 5) were tested over 23 calendar days with 2 replicates per sample per plate in a total of 54 runs with a randomized plate map. The total number of measurements was 108 per sample.

The values in the mean TREC and KREC copies/10 $^5$ cells column are transformed from logarithmic (Ln) mean values, and therefore, they represent geometric means in the copies/10 $^5$ 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 (Table 10 and Table 12). Summary mean, min and max copies/10 $^5$ cells, and SD and %CV results without log transformation for total imprecision are shown in Table 11 and Table 13.

Table 10. TREC precision data across three kit lots and three instruments

| Sample | Geometric Mean (Copies 105/cells) | Mean Ln (Copies 105/cells) | Repeata-bility 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 |
| 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 |

K203035 - Page 13 of 43

{13}

Table 11. TREC total variation results without logarithmic transformation

| | | Calculations without logarithmic transformation | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | N | Mean (Copies/ 105cells) | Min (Copies/ 105cells) | Max (Copies/ 105cells) | SD (Copies/ 105cells) | 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 | |

Table 12. KREC precision data across three kit lots and three instruments

| Sample | Geometric Mean (Copies 105/cells) | Mean Ln (Copies 105/cells) | Repeata-bility Ln SD | Between Run Ln SD | Between Instrument Ln 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 |

K203035 - Page 14 of 43

{14}

Table 13. KREC total variation results without logarithmic transformation

| | | Calculations without logarithmic transformation | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | N | Mean (Copies/ 105cells) | Min (Copies/ 105cells) | Max (Copies/ 105cells) | SD (Copies/ 105cells) | 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 | |

All 84 plates from both studies had results of the Eonis SCID-SMA Kit Controls within the acceptance limits. The data validity-check for each well resulted in a total of 15 out of 3354 well exclusions from the analysis (sample invalid rate is $0.4\%$ ). The precision of the device control kits is shown in Table 14a and the internal control RPP30 gene in Table 14b below.

Table 14a. Descriptive statistics of device kit control results (Ct values) at one site

| Analyte | Sample | N | N Ct N/A | Ct Mean | Ct SD | Ct CV% | Ct Min | Ct Max |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| KREC | C1 | 20 | 20 | N/A | N/A | N/A | NaN | NaN |
| | C2 | 20 | 0 | 33.5 | 0.56 | 1.68 | 32.3 | 34.6 |
| | C3 | 20 | 0 | 29.5 | 0.55 | 1.87 | 28.8 | 31.1 |
| | NTC | 20 | 20 | N/A | N/A | N/A | NaN | NaN |
| KREC ΔCt | C1 | 20 | 20 | N/A | N/A | N/A | NaN | NaN |
| | C2 | 20 | 0 | 33.5 | 0.56 | 1.68 | 32.3 | 34.6 |
| | C3 | 20 | 0 | 29.5 | 0.55 | 1.87 | 28.8 | 31.1 |
| | NTC | 20 | 20 | N/A | N/A | N/A | NaN | NaN |

K203035 - Page 15 of 43

{15}

| Analyte | Sample | N | N Ct N/A | Ct Mean | Ct SD | Ct CV% | Ct Min | Ct Max |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | C2 | 20 | 0 | 10.1 | 0.53 | 5.22 | 8.98 | 11.2 |
| | C3 | 20 | 0 | 6.07 | 0.43 | 7.10 | 5.48 | 7.20 |
| | NTC | 20 | 20 | N/A | N/A | N/A | NaN | NaN |
| RPP30 | C1 | 20 | 0 | 22.2 | 0.49 | 2.23 | 21.5 | 23.1 |
| | C2 | 20 | 0 | 21.7 | 0.37 | 1.69 | 21.1 | 22.4 |
| | C3 | 20 | 0 | 21.9 | 0.52 | 2.35 | 21.1 | 23.4 |
| | NTC | 20 | 19 | 37.5 | NA | N/A | 37.5 | 37.5 |
| TREC | C1 | 20 | 20 | N/A | N/A | N/A | NaN | NaN |
| | C2 | 20 | 0 | 33.4 | 0.77 | 2.31 | 32.3 | 35.4 |
| | C3 | 20 | 0 | 28.6 | 0.64 | 2.25 | 27.7 | 30.5 |
| | NTC | 20 | 20 | N/A | N/A | N/A | NaN | NaN |
| TREC ΔCt | C1 | 20 | 20 | N/A | N/A | N/A | NaN | NaN |
| | C2 | 20 | 0 | 33.4 | 0.77 | 2.31 | 32.3 | 35.4 |
| | C3 | 20 | 0 | 28.6 | 0.64 | 2.25 | 27.7 | 30.5 |
| | NTC | 20 | 20 | N/A | N/A | N/A | NaN | NaN |

N/A not applicable; NaN = Not a number (i.e., no Ct value generated)

Table 14b. RPP30 precision analysis on Ct values

| Sample | N | Mean | Within Run | | Between Run | | Between Instrument | | Within Kit lot | | Between Kit lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 107 | 23.4 | 0.70 | 3.0 | 0.39 | 1.7 | 0.30 | 1.3 | 0.42 | 1.8 | 0.86 | 3.7 | 0.96 | 4.1 |
| 2 | 108 | 23.2 | 0.54 | 2.3 | 0.44 | 1.9 | 0.23 | 1.0 | 0.34 | 1.5 | 0.73 | 3.2 | 0.81 | 3.5 |
| 3 | 106 | 23.2 | 0.69 | 3.0 | 0.25 | 1.1 | 0.09 | 0.4 | 0.34 | 1.5 | 0.74 | 3.2 | 0.81 | 3.5 |
| 4 | 108 | 24.2 | 0.99 | 4.1 | 0.33 | 1.4 | 0.36 | 1.5 | 0.45 | 1.9 | 1.11 | 4.6 | 1.20 | 4.9 |
| 5 | 106 | 24.0 | 0.79 | 3.3 | 0.00 | 0.0 | 0.22 | 0.9 | 0.54 | 2.2 | 0.82 | 3.4 | 0.98 | 4.1 |
| 6 | 107 | 24.4 | 0.59 | 2.4 | 0.24 | 1.0 | 0.00 | 0.0 | 0.70 | 2.9 | 0.64 | 2.6 | 0.95 | 3.9 |
| 7 | 107 | 24.2 | 1.17 | 4.8 | 0.00 | 0.0 | 0.19 | 0.8 | 0.64 | 2.7 | 1.19 | 4.9 | 1.35 | 5.6 |
| 8 | 107 | 24.4 | 0.81 | 3.3 | 0.00 | 0.0 | 0.20 | 0.8 | 0.70 | 2.9 | 0.84 | 3.4 | 1.09 | 4.5 |
| 9 | 106 | 25.6 | 0.69 | 2.7 | 0.00 | 0.0 | 0.00 | 0.0 | 0.37 | 1.4 | 0.69 | 2.7 | 0.78 | 3.0 |
| 10 | 107 | 23.9 | 0.62 | 2.6 | 0.14 | 0.6 | 0.34 | 1.4 | 0.56 | 2.4 | 0.72 | 3.0 | 0.92 | 3.8 |
| 11 | 107 | 23.0 | 0.45 | 2.0 | 0.34 | 1.5 | 0.00 | 0.0 | 0.40 | 1.8 | 0.56 | 2.4 | 0.69 | 3.0 |
| 12 | 107 | 23.7 | 0.81 | 3.4 | 0.28 | 1.2 | 0.25 | 1.1 | 0.55 | 2.3 | 0.89 | 3.8 | 1.05 | 4.4 |
| 13 | 106 | 24.5 | 0.81 | 3.3 | 0.50 | 2.1 | 0.2 | 0.8 | 0.72 | 2.9 | 0.97 | 4.0 | 1.21 | 5.0 |

Qualitative imprecision was determined in accordance with CLSI document EP12-A2 to classify the results, using the screening performance study cut-offs (262 copies/10 $^5$ cells for TREC, 484 copies/10 $^5$ cells for KREC). The C5-C95 interval was determined to be 79-626 copies/10 $^5$ cells for TREC, and 189-1064 copies/10 $^5$ cells for KREC. Concentrations outside of these intervals were consistently analyte negative (concentrations $&lt;\mathrm{C}5$ , equals to screen positive) or consistently analyte positive (concentrations $&gt;\mathrm{C}95$ , equals to screen negative).

K203035 - Page 16 of 43

{16}

# 2. Linearity:

Linearity was determined with 1 kit lot and 1 operator. Three (3) sets of contrived samples were used for this evaluation and are described below. The samples were diluted to 9 levels with 4 replicates for each level. Samples were punched at 4 replicates using a DBS puncher and then transferred into a new 96-well plate in a randomized order and were extracted and run using JANUS automated liquid handlers. Acceptance criteria were that all analytes, TREC, KREC and RPP30, must have PCR efficiency between 80% and 120% with a coefficient of determination, denoted R² ≥ 95% within their measuring ranges. The acceptance criteria for linearity for TREC and KREC concentrations were, on their measuring ranges, the maximal difference of the best fitting model from the linear model must be ± 25% (calculated in copies/10⁵ cells).

## Sample Set 1: Estimation of PCR Efficiency for TREC, KREC and RPP30

The first set of samples were prepared by spiking leucocyte-depleted human red blood cells, which had the hematocrit adjusted to 51.9, with linearized TREC and KREC plasmids and cell lines covering the required measuring ranges (TREC: 52 to 7800 copies/μL blood; KREC: 67 to 10,100 copies/μL blood; RPP30: 2670 to 400,000 copies/μL blood, per spiked cell numbers), a semi-log linear regression line plot of Ct value vs. log of input nucleic acid in the unit of copies/μL blood was first analyzed for each individual analyte (TREC, KREC and RPP30) and the corresponding coefficient of determination (R²) of the linear regression of the mean Ct values and PCR efficiency of each analyte were calculated. As shown in Table 15, all 3 analytes had R² &gt; 99%, and their estimates of PCR efficiency were between 80% and 120%.

Table 15. Estimate of R² and PCR efficiency of TREC, KREC and RPP30

| Analyte Name | R² | PCR efficiency estimate | Lower 95% CL | Upper 95% CL |
| --- | --- | --- | --- | --- |
| TREC | 0.993 | 83.9% | 76.7% | 92.6% |
| KREC | 0.995 | 87.6% | 81.2% | 95.0% |
| RPP30 | 0.992 | 99.9% | 89.9% | 112.3% |

## Sample Set 2 and 3: Estimation of Linearity of the TREC and KREC Assay

Linearity was determined in accordance with CLSI document EP06-A2 and was designed to evaluate the linearity of TREC and KREC in the unit of copies/10⁵ cells calculated using ΔCt TREC and ΔCt KREC for each dilution. A high concentration sample was prepared by resuspending WBC (White Blood Cells) pellets concentrated from the 60 mL of human umbilical cord bloods into 1 mL of human umbilical cord blood to achieve TREC and KREC Ct close to the lowest values (i.e., high concentrations) observed in a population of newborn specimens (28.34 for TREC and 26.91 for KREC). Samples were then diluted with leukocyte depleted whole blood into nine levels of serial dilutions down to a level close to their LoQs (242 copies/105 cells for TREC and 459 copies/105 cells for KREC). In addition, a third set of samples were created to provide additional information on the KREC linearity at lower KREC concentrations because the second set did not cover the measuring range. This low concentration sample set 3 was prepared by diluting umbilical cord blood with

K203035 - Page 17 of 43

{17}

leucocyte-depleted human red blood. HL-60 cells were spiked into the leucocyte-depleted blood to adjust the RPP30 Ct values to match the RPP30 Ct value in cord blood. Using the observed value for undiluted sample and the consecutive dilution factors, the expected values at copies/10 $^5$ cells scale were calculated. Linearity of TREC and KREC was then determined by fitting regression of Ln Measured vs. Ln Expected value. Weights for regression fit were calculated from the observed variability of each level. Using the obtained equation, the predicted values at each dilution level and the deviations between measured and predicted values were calculated. The descriptive statistics of each linearity sample level are presented in Table 16 and Table 17. The data for KREC represents the combined datasets. The measured results and fitted models are shown in Figure 1 and 3 the relative deviations in Figures 2 and 4. For TREC, all points were within the study acceptance criteria of $\pm 25\%$ , with highest relative deviation of $-14.3\%$ . For KREC, all points were within the study acceptance criteria of $\pm 25\%$ , with highest relative deviation of $17.3\%$ .

Table 16. Descriptive statistics and calculated weights - TREC

| RC* | N | Copies/105Mean | Ln CopiesMean | Copies/105SD | Ln CopiesSD | Weight | Copies/105Min | Copies/105Max |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.000 | 4 | 4316 | 8.37 | 461 | 0.11 | 331 | 3790 | 4890 |
| 0.690 | 4 | 2441 | 7.80 | 834 | 0.37 | 29.2 | 1430 | 3330 |
| 0.476 | 3 | 1588 | 7.37 | 459 | 0.31 | 31.2 | 1110 | 1910 |
| 0.328 | 4 | 1353 | 7.21 | 257 | 0.19 | 111 | 1100 | 1710 |
| 0.226 | 4 | 1287 | 7.16 | 260 | 0.20 | 100 | 999 | 1630 |
| 0.156 | 4 | 508 | 6.23 | 148 | 0.34 | 34.6 | 309 | 630 |
| 0.108 | 4 | 185 | 5.22 | 113 | 0.71 | 7.93 | 66.5 | 340 |
| 0.074 | 3 | 114 | 4.74 | 64.5 | 0.48 | 13.0 | 82.8 | 198 |
| 0.051 | 4 | 93.7 | 4.54 | 77.8 | 0.78 | 6.57 | 40.8 | 190 |

*Relative concentration

Figure 1. Fitted linear model TREC

K203035 - Page 18 of 43

{18}

Figure 2. Relative deviations and the study limits - TREC

Table 17. Descriptive statistics and calculated weights - KREC

| RC* | Sample | N | Hit rate | Copies/105cells Mean | Ln Copies Mean | Ln Copies SD | Weight | Copies/105cells Min | Copies/105cells Max |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1.000 | Lin | 4 | 1 | 24343 | 10.1 | 0.14 | 204 | 19700 | 26800 |
| 0.690 | Lin | 4 | 1 | 15835 | 9.67 | 0.19 | 111 | 12500 | 19500 |
| 0.476 | Lin | 3* | 1 | 10405 | 9.25 | 0.03 | 3330 | 10100 | 10800 |
| 0.328 | Lin | 4 | 1 | 8519 | 9.05 | 0.05 | 1600 | 7990 | 9020 |
| 0.226 | Lin | 4 | 1 | 6768 | 8.82 | 0.08 | 625 | 6290 | 7470 |
| 1.000 | L2 | 20 | 1 | 6438 | 8.77 | 0.26 | 296 | 4170 | 11500 |
| | L3 | 20 | 1 | 5064 | 8.53 | 0.29 | 238 | 2380 | 8880 |
| | L1 | 20 | 1 | 3905 | 8.27 | 0.36 | 154 | 2500 | 8160 |
| 0.156 | Lin | 4 | 1 | 3533 | 8.17 | 0.45 | 19.8 | 2170 | 5800 |
| 0.700 | L1 | 20 | 1 | 3072 | 8.03 | 0.33 | 184 | 1440 | 5130 |
| 0.108 | Lin | 4 | 1 | 2836 | 7.95 | 0.34 | 34.6 | 1720 | 3750 |
| 0.600 | L1 | 20 | 1 | 2644 | 7.88 | 0.32 | 195 | 1500 | 4650 |
| 0.074 | Lin | 3* | 1 | 2490 | 7.82 | 0.16 | 117 | 2090 | 2790 |
| 0.500 | L1 | 20 | 1 | 2080 | 7.64 | 0.43 | 108 | 567 | 4320 |
| 0.300 | L3 | 20 | 1 | 1588 | 7.37 | 0.50 | 80.0 | 375 | 3540 |
| 0.051 | Lin | 4 | 1 | 1249 | 7.13 | 0.37 | 29.2 | 780 | 1910 |
| 0.200 | L1 | 19 | 0.95 | 1188 | 7.08 | 0.36 | 147 | 619 | 2200 |
| 0.150 | L2 | 20 | 1 | 871 | 6.77 | 0.53 | 71.2 | 356 | 2850 |
| | L3 | 20 | 1 | 804 | 6.69 | 0.64 | 48.8 | 82.9 | 1570 |
| 0.100 | L2 | 19 | 0.95 | 757 | 6.63 | 0.47 | 86.0 | 344 | 2970 |
| 0.080 | L2 | 20 | 1 | 478 | 6.17 | 0.92 | 23.6 | 16.2 | 1580 |
| 0.040 | L2 | 14 | 0.7 | 305 | 5.72 | 1.03 | 13.2 | 54.9 | 1030 |
| 0.080 | L3 | 18 | 0.9 | 296 | 5.69 | 0.68 | 38.9 | 74.1 | 936 |
| | L1 | 16 | 0.8 | 255 | 5.54 | 0.63 | 40.3 | 75.2 | 620 |

K203035 - Page 19 of 43

{19}

| RC* | Sample | N | Hit rate | Copies/105cells Mean | Ln Copies Mean | Ln Copies SD | Weight | Copies/105cells Min | Copies/105cells Max |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0.030 | L2 | 13 | 0.65 | 226 | 5.42 | 0.68 | 28.1 | 52.9 | 480 |
| 0.020 | L3 | 4 | 1 | 200 | 5.30 | 0.48 | 17.4 | 152 | 411 |
| 0.060 | L3 | 18 | 0.9 | 187 | 5.23 | 0.97 | 19.1 | 29.8 | 804 |
| 0.030 | L3 | 14 | 0.7 | 178 | 5.18 | 0.57 | 43.1 | 75.7 | 407 |
| 0.010 | L3 | 9 | 0.45 | 174 | 5.16 | 0.64 | 22.0 | 80.1 | 608 |
| 0.040 | L3 | 17 | 0.85 | 166 | 5.11 | 1.03 | 16.0 | 10.5 | 392 |
| 0.020 | L2 | 13 | 0.65 | 151 | 5.02 | 0.90 | 16.0 | 22.2 | 502 |
| 0.010 | L2 | 9 | 0.45 | 147 | 4.99 | 0.34 | 77.9 | 61.7 | 190 |
| 0.030 | L1 | 13 | 0.65 | 129 | 4.86 | 0.65 | 30.8 | 44.3 | 466 |
| 0.010 | L1 | 3 | 0.15 | 117 | 4.76 | 0.56 | 9.57 | 61.8 | 166 |

*Relative Concentration

Figure 3. Fitted linear model - KREC

Figure 4. Relative deviations and the study limits - KREC

K203035 - Page 20 of 43

{20}

The TREC analyte is demonstrated to be linear from 94 copies/10⁵ cells to 4316 copies/10⁵ cells with observed maximum deviation of -14.3%. The KREC analyte is demonstrated to be linear from 117 to 24343 copies/10⁵ cells with observed maximum deviation of 17.3%.

## 3. Analytical Specificity

### (i) Interference

The Eonis SCID-SMA kit was evaluated for interference from the following potential endogenous and exogenous sources:

- Hemoglobin up to 200g/L
- Conjugated bilirubin up to 16.6 mg/dL (166mg/L)
- Unconjugated bilirubin up to 10mg/dL (100mg/L)
- Intralipid up to 1500 mg/dL (15mg/mL)
- Li-hep up to 7500 USP/dL
- Na Citrate up to 0.0645 mol/L
- EDTA up to 9.8 mg/mL

The interference study was performed by 3 operators using 1 Eonis SCID-SMA Kit and 3 QuantStudio Dx Real-time PCR instruments. Three levels of TREC and KREC target DNA (normal, near cut-off and endogenous were tested in conjunction with each level of interferent (low and high). Blood samples were collected from healthy individuals and the hematocrit was adjusted to 40-55% corresponding to the hematocrit of neonates (except blood used in the hemoglobin sample series). Blood samples were then spiked with TREC and KREC plasmid DNA to obtain the desired concentration levels. Blood samples with the added potential interferent were used to prepare dried blood spots. The blood spots were dried overnight and stored at -80°C. The specimens used are shown in Table 18.

Table 18. Samples used in the interference study

| Sample Name (Abbreviation) | TREC Level | KREC Level | Interferent Substance Level |
| --- | --- | --- | --- |
| Normal Targets+ Low Interferent (NL) | Normal | Normal | Low (0%) |
| Normal Targets+ High Interferent (NH) | Normal | Normal | High (100%) |
| Cut-off Targets + Low Interferent (CL) | Near Cut-off | Near Cut-off | Low (0%) |
| Cut-off Targets + High Interferent (CH) | Near Cut-off | Near Cut-off | High (100%) |
| Endogenous Targets + Low Interferent (EL) | Endogenous | Endogenous | Low (0%) |
| Endogenous Targets + High Interferent (EH) | Endogenous | Endogenous | High (100%) |

The study generated a total of 544 sample results not including controls (7 interfering substances x 2 interferent levels x 3 target DNA levels x 13 repeats). Acceptance criteria

K203035 - Page 21 of 43

{21}

were based on observed imprecision in the precision studies. The results indicated there was no interference observed for any of the tested interferents. Hemoglobin at up to 200 g/L, conjugated bilirubin at up to 16.6 mg/dL (=166 mg/L), unconjugated bilirubin at up to 10 mg/dL (=100 mg/L), Intralipid at up to 1500 mg/dL (15 mg/mL), Li-heparin at up to 7500 USP/dL, Na-citrate at up to 0.0645 mol/L, and EDTA at up to 9.8 mg/mL did not significantly interfere with the Eonis SCID-SMA kit.

(ii) Primer and Probe Specificity

An in silico study was performed to assess the specificity of the primers and probes used in the Eonis assay. Primer and probe specificity was assessed by using the online Standard Nucleotide BLAST tool (Optimize for highly similar sequences, megablast) against the Human genomic plus transcript (Human G + T) database published by the National Center for Biotechnology Information (NCBI).

The BLAST analysis confirmed that there were only hits with 100% homology to the T receptor alpha delta locus (TREC) on chromosome 14 along, the full length of the primer and probe sequences, and 100% homology to the immunoglobulin kappa locus (IGK) on chromosome 2, and to the ribonuclease P subunit p30 locus (RPP30) on chromosome 10. There was no significant homology to other genome sequences.

4. Assay Reportable Range:

Refer to the Results interpretation section.

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

(i) Traceability

There are no recognized standards or reference material for TREC or KREC DNA. The kit contains controls employed in the determination of TREC and KREC values.

(ii) Specimen Stability

The influence of storage time, temperature, and humidity was studied using DBS samples prepared with two brands of filter paper. Samples were prepared by mixing cell lines and red blood cell concentrate (mimicking a true positive sample for all 3 conditions), using cord blood (mimicking a true negative sample) or by mixing cell lines, red blood cell concentrate and cord blood for the near cut-off level samples.

After collection and preparation, the blood spots were dried overnight at ambient temperature (+19--+25°C). The dried blood spots were then stored at different storage conditions. Storage of specimens in an environment with elevated temperatures and humidity increases the risk of inaccurate TREC/KREC results in normal or near cut-off samples. The TREC/KREC results are shown in Figure 5a and Figure 5b.

K203035 - Page 22 of 43

{22}

K203035 - Page 23 of 43

TREC Sample 1 (Copies/10⁵ cells), Filter paper brand 1

TREC Sample 2 (Copies/10⁵ cells), Filter paper brand 1

TREC Sample 3 (Copies/10⁵ cells), Filter paper brand 2

TREC Sample 4 (Copies/10⁵ cells), Filter paper brand 1

{23}

Figure 5a. TREC copies across different storage conditions in samples mimicking true positive (Sample 1), true negative (Sample 4) and in-between (Sample 2 and Sample 3)



K203035 - Page 24 of 43

{24}

Figure 5b. KREC copies across different storage conditions in samples mimicking true positive (Sample 1), true negative (Sample 4) and in-between (Sample 2 and Sample 3)

# (iii) Reagent Stability:

Stability studies were conducted to demonstrate the real time, in-use, and on-board stability as well as transport shipping stress simulation of the Eonis DNA Extraction Kit components and the Eonis SCID-SMA kit. The tests were performed after a transport simulation treatment that simulates the worst-case conditions that can occur during packaging and shipping. When a transport simulation treatment was performed, all the kit components were used in the study. The stability testing was conducted in accordance with guideline EN ISO 23640 In vitro diagnostic medical devices - Evaluation of stability of in vitro diagnostic reagents (2015) and CLSI Guideline EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents, Approved Guideline- Volume 29, No. 20 (2009).

The testing was performed at 7 months of the components' shelf life. All Mean Ct values and variation (standard deviation) results fulfilled the acceptance criteria at all tested time points. At all tested time points, the results passed the data validity check. (Refer to DEN200044 for details of the study design.) The following stability conditions apply based on the results:

- Eonis DNA Extraction Kit components (Wash Solution and Elution Solution) are stable for 14 days at $+19 - +25^{\circ}\mathrm{C}$ after first use.
- Eonis DNA extraction Kit components are stable in troughs for 30 minutes at $+19 - +25^{\circ}\mathrm{C}$ .
Note: The Eonis DNA extraction Kit components are stable on board the JANUS Extraction instrument in troughs for 2 hours.
- PCR Reagents 1 and 2 once thawed from $-30^{\circ}\mathrm{C}$ to $-16^{\circ}\mathrm{C}$ , are stable for 14 days at $+2^{\circ}\mathrm{C}$ to $+8^{\circ}\mathrm{C}$ .
- PCR Reagents 1 and 2 are stable in their tubes uncapped on-board the JANUS PCR Mastermix instrument for 2 hours at $+19$ to $+25^{\circ}\mathrm{C}$ .
- The mixture of PCR Reagents 1 and 2 is stable for 15 minutes in a trough at $+19$ to $+25^{\circ}\mathrm{C}$ .
- The mixture of PCR Reagents 1 and 2 is stable for 2 hours in a sealed plate protected from light.
- The SCID-SMA Kit controls are stable for 14 days after first use when stored at $-30^{\circ}\mathrm{C}$ to $-16^{\circ}\mathrm{C}$ in a sealed bag with desiccant.

K203035 - Page 25 of 43

{25}

- The SCID-SMA Kit controls are stable on-board the JANUS Extraction instrument at +19 to +25°C for 4 hours after they are punched into 96-well plates.
- Based on the results of the real-time and transport simulation stability study a shelf life 180 days (6 months) is claimed for all Eonis SCID-SMA kit components, when stored at the labeled storage temperature of -30°C - -16°C.

6. Detection Limit:

(i) Limit of Blank (LoB)

The LoB is the highest measurement result that is likely to be observed for a blank sample that contains no analyte. The LoB study was performed using contrived analyte negative samples created by spiking a Coriell cell line into leukocyte depleted blood which was TREC and KREC negative. Five (5) individual contrived analyte-negative samples were prepared by spiking the Coriell cell line into 5 different lots of leukocyte-depleted human blood with the hematocrit adjusted to 40-55%. The study was performed by 1 operator using 2 sets of Instrument Systems and 2 kit lots run over 5 days for a total of 10 runs. Six replicates were tested in each run totaling 300 results (150 results/kit lot). For the RPP30 LoB, blank samples were used (no punch/no template DNA), 20 replicates were tested in each run totaling 200 results (100 results/kit lot). Each plate contained kit controls NTC, C1, C2 and C3, assayed in duplicate. Acceptance criteria were that the LoB of RPP30 must be zero; or any samples that have RPP30 Ct ≥ 32.00 will be reported as 'Invalid' for TREC and KREC. The LoB of TREC and KREC must be no greater than the LoD.

For TREC and KREC, all of the replicates had no Ct value reported. For RPP30, there were two replicates having Ct values reported at 34.49 and 36.23; however, as this would lead to an invalid result for the sample, the percentage of false-positive results for RPP30, defined as the percentage of replicates of the blank sample that had a valid RPP30 Ct value ≤ 32.00 was also zero for both kit lots. The LoB of TREC is 0 copies/μL blood, and the LoB of KREC is 0 copies/μL blood.

(ii) Limit of Detection (LoD) and Limit of Quantitation (LoQ)

LoD and LoQ were evaluated separately for TREC and KREC. The LoD and LoQ studies were performed using contrived samples prepared by diluting cord blood with adult leucocyte-depleted human red blood that had TREC and/or KREC levels that were undetectable by the Eonis SCID-SMA Kit. HL-60 cells were spiked into the adult leucocyte-depleted blood to adjust the RPP30 Ct values to match the RPP30 Ct value in cord blood (within 1 Ct difference). Each cord blood sample was diluted into different levels in which at least the lowest three levels had yield hit rates within the range of 0.10 to 0.90 using the corresponding diluents. The samples were then spotted onto filter paper and dried overnight. For each analyte, TREC or KREC, the study was performed using 2 sets of instruments and 2 kit lots. One operator performed 2 runs per day, for 4 days, totaling 8 runs.

For each sample and each dilution, including negative samples (diluents), 5 replicates were tested in each run, totaling 960 results with 20 replicates per dilution per lot. The final dilutions used in the study and the corresponding number of positive results observed, total

K203035 - Page 26 of 43

{26}

number of measurements and hit rates observed for TREC and KREC are summarized in Tables 19 and 20, respectively.

Table 19. TREC calculated hit rate percentages with sample concentrations in copies/105 cells

| Kit Lot | Cord Blood Sample | Sample Level | Dilution | Concentration in copies/105cells | Observed Positives | Observed Negatives | Hit Rate % |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | L1 | Cord Blood | 1.00 | 973 | 0 | 20 | 100% |
| | | Level 1 | 2.00 | 528 | 0 | 20 | 100% |
| | | Level 2 | 3.33 | 428 | 0 | 20 | 100% |
| | | Level 3 | 5.00 | 137 | 0 | 20 | 100% |
| | | Level 4 | 10.0 | 77 | 5 | 15 | 75% |
| | | Level 5 | 12.5 | 44 | 6 | 14 | 70% |
| | | Level 6 | 16.7 | 44 | 9 | 11 | 55% |
| | | Level 7 | 25.0 | 36 | 12 | 8 | 40% |
| | | Diluent | N/A | 21 | 18 | 2 | 10% |
| | L2 | Cord Blood | 1.00 | 2670 | 0 | 20 | 100% |
| | | Level 1 | 6.67 | 237 | 0 | 20 | 100% |
| | | Level 2 | 10.0 | 166 | 2 | 18 | 90% |
| | | Level 3 | 12.5 | 122 | 2 | 18 | 90% |
| | | Level 4 | 25.0 | 50 | 11 | 9 | 45% |
| | | Level 5 | 33.3 | 52 | 10 | 10 | 50% |
| | | Level 6 | 50.0 | 38 | 13 | 7 | 35% |
| | | Level 7 | 100 | 15 | 17 | 3 | 15% |
| | | Diluent | N/A | 0 | 20 | 0 | 0% |
| | L3 | Cord Blood | 1.00 | 1525 | 0 | 20 | 100% |
| | | Level 1 | 3.33 | 365 | 0 | 20 | 100% |
| | | Level 2 | 6.67 | 194 | 2 | 18 | 90% |
| | | Level 3 | 12.5 | 83 | 3 | 17 | 85% |
| | | Level 4 | 16.7 | 44 | 7 | 13 | 65% |
| | | Level 5 | 25.0 | 39 | 5 | 15 | 75% |
| | | Level 6 | 33.3 | 55 | 9 | 11 | 55% |
| | | Level 7 | 50.0 | 17 | 14 | 6 | 30% |
| Level 8 | | 100 | 22 | 16 | 4 | 20% | |
| Diluent | | N/A | 0 | 20 | 0 | 0% | |
| 2 | L1 | Cord Blood | 1.00 | 1141 | 0 | 20 | 100% |
| | | Level 1 | 2.00 | 395 | 0 | 20 | 100% |

K203035 - Page 27 of 43

{27}

Table 20. KREC calculated hit rate percentages with sample concentrations in copies/10$^5$ cells

| Kit Lot | Cord Blood Sample | Sample Level | Dilution | Concentration in copies/10$^5$ cells | Observed Positives | Observed Negatives | Hit Rate % |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Level 2 | 3.33 | 596 | 0 | 20 | 100% |
| | | Level 3 | 5.00 | 106 | 1 | 19 | 95% |
| | | Level 4 | 10.0 | 63 | 7 | 13 | 65% |
| | | Level 5 | 12.5 | 84 | 16 | 4 | 20% |
| | | Level 6 | 16.7 | 32 | 9 | 11 | 55% |
| | | Level 7 | 25.0 | 17 | 17 | 3 | 15% |
| | | Diluent | N/A | 45 | 19 | 1 | 5% |
| | L2 | Cord Blood | 1.00 | 2490 | 0 | 20 | 100% |
| | | Level 1 | 6.67 | 240 | 0 | 20 | 100% |
| | | Level 2 | 10.0 | 118 | 3 | 17 | 85% |
| | | Level 3 | 12.5 | 109 | 2 | 18 | 90% |
| | | Level 4 | 25.0 | 54 | 8 | 12 | 60% |
| | | Level 5 | 33.3 | 30 | 6 | 14 | 70% |
| | | Level 6 | 50.0 | 25 | 11 | 9 | 45% |
| | | Level 7 | 100 | 29 | 17 | 3 | 15% |
| | | Diluent | N/A | 0 | 20 | 0 | 0% |
| | L3 | Cord Blood | 1.00 | 1510 | 0 | 20 | 100% |
| | | Level 1 | 3.33 | 321 | 2 | 18 | 90% |
| | | Level 2 | 6.67 | 136 | 0 | 20 | 100% |
| | | Level 3 | 12.5 | 72 | 1 | 19 | 95% |
| | | Level 4 | 16.7 | 59 | 6 | 14 | 70% |
| | | Level 5 | 25.0 | 34 | 11 | 9 | 45% |
| | | Level 6 | 33.3 | 19 | 13 | 7 | 35% |
| | | Level 7 | 50.0 | 44 | 15 | 5 | 25% |
| Level 8 | | 100 | 34 | 17 | 3 | 15% | |
| Diluent | | N/A | 0 | 20 | 0 | 0% | |

K203035 - Page 28 of 43

{28}

K203035 - Page 29 of 43
| Kit Lot | Cord Blood Sample | Sample Level | Dilution | Concentration in copies/10^{5} cells | Observed Positives | Observed Negatives | Hit Rate % |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Level 3 | 2.00 | 2080 | 0 | 20 | 100% |
| | | Level 4 | 5.00 | 1188 | 0 | 19 | 100% |
| | | Level 5 | 12.5 | 255 | 4 | 16 | 80% |
| | | Level 6 | 33.3 | 129 | 7 | 13 | 65% |
| | | Level 7 | 100 | 117 | 17 | 3 | 15% |
| | | Diluent | N/A | 0 | 20 | 0 | 0% |
| | L2 | Cord Blood | 1.00 | 6438 | 0 | 20 | 100% |
| | | Level 1 | 6.67 | 871 | 0 | 20 | 100% |
| | | Level 2 | 10.0 | 757 | 0 | 19 | 100% |
| | | Level 3 | 12.5 | 478 | 0 | 20 | 100% |
| | | Level 4 | 25.0 | 305 | 6 | 14 | 70% |
| | | Level 5 | 33.3 | 226 | 7 | 13 | 65% |
| | | Level 6 | 50.0 | 151 | 7 | 13 | 65% |
| | | Level 7 | 100 | 147 | 11 | 9 | 45% |
| | | Diluent | N/A | 0 | 20 | 0 | 0% |
| | L3 | Cord Blood | 1.00 | 5064 | 0 | 20 | 100% |
| | | Level 1 | 3.33 | 1588 | 0 | 20 | 100% |
| | | Level 2 | 6.67 | 804 | 0 | 20 | 100% |
| | | Level 3 | 12.5 | 296 | 2 | 18 | 90% |
| | | Level 4 | 16.7 | 187 | 2 | 18 | 90% |
| | | Level 5 | 25.0 | 166 | 3 | 17 | 85% |
| Level 6 | | 33.3 | 178 | 5 | 14 | 74% | |
| Level 7 | | 50.0 | 200 | 16 | 4 | 20% | |
| Level 8 | | 100 | 174 | 11 | 9 | 45% | |
| Diluent | | N/A | 0 | 20 | 0 | 0% | |
| 2 | L1 | Cord Blood | 1.00 | 4064 | 0 | 20 | 100% |
| | | Level 1 | 1.43 | 4146 | 0 | 20 | 100% |
| | | Level 2 | 1.67 | 3828 | 0 | 20 | 100% |
| | | Level 3 | 2.00 | 2039 | 0 | 20 | 100% |
| | | Level 4 | 5.00 | 846 | 0 | 20 | 100% |
| | | Level 5 | 12.5 | 209 | 2 | 18 | 90% |
| | | Level 6 | 33.3 | 191 | 6 | 14 | 70% |
| | | Level 7 | 100 | 128 | 15 | 5 | 25% |
| | | Diluent | N/A | 0 | 20 | 0 | 0% |
| | L2 | Cord Blood | 1.00 | 5825 | 0 | 20 | 100% |

{29}

| Kit Lot | Cord Blood Sample | Sample Level | Dilution | Concentration in copies/10^{5} cells | Observed Positives | Observed Negatives | Hit Rate % |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Level 1 | 6.67 | 1064 | 0 | 20 | 100% |
| | | Level 2 | 10.0 | 556 | 0 | 20 | 100% |
| | | Level 3 | 12.5 | 412 | 0 | 20 | 100% |
| | | Level 4 | 25.0 | 235 | 1 | 19 | 95% |
| | | Level 5 | 33.3 | 245 | 3 | 17 | 85% |
| | | Level 6 | 50.0 | 116 | 7 | 13 | 65% |
| | | Level 7 | 100 | 134 | 10 | 10 | 50% |
| | | Diluent | N/A | 0 | 20 | 0 | 0% |
| | L3 | Cord Blood | 1.00 | 4915 | 0 | 20 | 100% |
| | | Level 1 | 3.33 | 1510 | 0 | 20 | 100% |
| | | Level 2 | 6.67 | 659 | 0 | 20 | 100% |
| | | Level 3 | 12.5 | 311 | 2 | 18 | 90% |
| | | Level 4 | 16.7 | 287 | 3 | 17 | 85% |
| | | Level 5 | 25.0 | 296 | 3 | 17 | 85% |
| | | Level 6 | 33.3 | 167 | 5 | 15 | 75% |
| | | Level 7 | 50.0 | 158 | 9 | 11 | 55% |
| | | Level 8 | 100 | 143 | 15 | 5 | 25% |
| | | Diluent | N/A | 0 | 20 | 0 | 0% |

The LoD was calculated using Probit analysis at 95% probability for each reagent kit lot. Probit analysis in copies/10⁵ cells unit was performed. The data collected from all 3 cord blood samples and their dilutions were pooled for the calculation. The results for both kit lots with 95% confidence intervals for TREC and KREC are summarized in Tables 21 and 22, respectively. The LoD estimate is the concentration value that Probit analysis gives an estimated hit rate of 95%.

Table 21. TREC LoD estimates based on Probit analysis with hit rate percentages and sample concentrations in copies/10⁵ cells unit

| Kit lot | LoD estimate (copies/10⁵ cells) | 95% confidence interval |
| --- | --- | --- |
| 1 | 174 | 137 - 242 |
| 2 | 242 | 184 - 360 |

K203035 - Page 30 of 43

{30}

Table 22. KREC LoD estimates based on Probit analysis with hit rate percentages and sample concentrations in copies/10⁵ cells unit

| Kit lot | LoD estimate (copies/10⁵ cells) | 95% confidence interval |
| --- | --- | --- |
| 1 | 459 | 371 - 669 |
| 2 | 340 | 292 - 439 |

The LoQ was evaluated as the functional sensitivity that represents the measurand concentration associated with a desired within-laboratory precision, as exemplified by CLSI guideline EP17-A2 Appendix D. Only the dilutions that yielded 100% hit rates were included in the LoQ calculation. For each qualified dilution, the mean and the SD of the concentrations in Ln (copies/10⁵ cells) were calculated. A power function model (SD vs. the mean concentration) was used to fit the datasets for each reagent kit lot. The LoQ estimate for each reagent kit lot was determined as the predicted lowest concentration that has within-laboratory precision equal to an SD ≤ 0.90 Ln (copies/10⁵ cells) for TREC and ≤ 1.49 Ln (copies/10⁵ cells) for KREC. The results are shown in Tables 23 and 24.

Table 23. TREC LoQ estimates based on conservative approach in copies/10⁵ cells

| Kit lot | LoD (copies/10⁵ cells) | LoQ estimate (copies/10⁵ cells) | Final LoQ (=LoD) (copies/10⁵ cells) |
| --- | --- | --- | --- |
| 1 | 174 | 30 | 242 |
| 2 | 242 | 123 | |

Table 24. KREC LoQ estimates based on conservative approach in copies/10⁵ cells

| Kit lot | LoD (copies/10⁵ cells) | LoQ estimate (copies/10⁵ cells) | Final LoQ (=LoD) (copies/10⁵ cells) |
| --- | --- | --- | --- |
| 1 | 459 | 7 | 459 |
| 2 | 340 | 0 | |

The following results were determined for LoD and LoQ of TREC and KREC:

$$
\mathrm{LoD} = \mathrm{LoQ}
$$

TREC: 242 copies/10⁵ cells

KREC: 459 copies/10⁵ cells

7. Assay Cut-Off:

Refer to the Clinical Cut-off in Section VII.D.

8. Accuracy (Instrument):

Not applicable

K203035 - Page 31 of 43

{31}

9. Carry-Over:

A study was conducted to evaluate the potential for sample carryover to demonstrate control of contamination during testing of samples with the Eonis reagent kits. The carryover test was performed using 1 kit lot. The high-analyte positive sample was prepared by spiking plasmid DNAs of each analyte into leukocyte-depleted human blood at levels lower than 29.71 for TREC Ct, 29.22 for KREC Ct, and 22.31 for RPP30 Ct, corresponding to the 10% quantiles of a population of newborn specimens. The high analyte-positive sample and the analyte-negative sample were placed in alternating rows and columns to produce a checkerboard configuration. The observed carryover percentage was 0% for the three analytes.

B Comparison Studies:

1. Method Comparison with Predicate Device:

Not applicable

2. Matrix Comparison:

Filter Paper Reproducibility

A filter paper reproducibility study was conducted using filter paper from 2 manufacturers in order to evaluate manufacturer difference, lot differences, as well as to assess homogeneity across dried blood spots (DBS). Between paper manufacturer and paper lots were evaluated using samples spotted on 3 lots of each filter paper brand.

The study was conducted using a subset of the samples that were used in the precision and 3-site reproducibility studies (although some at different concentrations) covering high, normal and low levels of each analyte (Table 5).

- Sample 1 High TREC: Prepared by spiking TREC plasmid into cord blood at 2000 copies/μL with hematocrit adjusted to 51.8 (approx. Ct value 28.19)
- Sample 2 High KREC: Prepared by spiking TREC plasmid into cord blood at 2000 copies/μL with hematocrit adjusted to 51.8 (approx. Ct value 28.65)
- Sample 3: Prepared by spotting cord blood with hematocrit adjusted to 51.8 onto 3 lots of each filter paper brand
- Sample 11: Prepared by spiking TREC plasmid at 700 copies/μL, KREC plasmid at 1000 copies/μL and Coriell cells at 15000 cells/ μL into leukocyte depleted blood with hematocrit adjusted to 49.
- Sample 12 Low levels (above cut-off) TREC and KREC: Prepared by diluting 400 μL of cord blood with hematocrit adjusted to 50.9 in 600 μL of adult whole blood with hematocrit adjusted to 52.9 (approx. TREC Ct value 33.29 and approx. KREC Ct value 32.20)
- Sample 13 Low levels (below cut-off) TREC and KREC: Prepared by spotting adult whole blood with hematocrit adjusted to 52.9 onto 3 lots of each paper brand (approx. TREC Ct value no Ct and approx. KREC Ct value 3.44)

K203035 - Page 32 of 43

{32}

Each analyte was manually spotted on 3 filter paper lots at 85 µL per spot.

One operator performed 1 run per day for 5 days for a total of 5 runs and using 1 kit lot and 1 Eonis SCID-SMA test system. Six samples were prepared on 3 lots of each filter paper brand for a total of 36 conditions (6 samples x 3 lots x 2 filter paper brands = 36). Each run consisted of 3 96-well extraction plates (2 randomized sample sets per plate plus 2 sets of kit controls) and 1 384-well PCR plate in each testing day.

To assess the homogeneity of the analytes across the DBS, 5 replicates of each condition were punched from 5 different locations of each spot in the following order: top, left, center, right, bottom. The study generated 900 results (25 replicates x 6 samples x 3 filter paper lots x 2 filter paper brands) with 150 results per sample and 450 results per filter paper brand. The total variation InSD results are compared to the product specification for total variation (“Acceptance criteria”) and are shown in Table 25. The total variation lognormal CV% results are presented in Table 26.

Table 25. TREC and KREC precision analysis with between variation estimates across paper manufacturers and across paper lots

| Analyte | Sample | N | Mean copies/10^{6} cells | Mean Ln copies/10^{6} cells | Within Paper Lot InSD | Between Paper Lot InSD | Between Manufacturer InSD | Total InSD | Specification |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TREC | 2 | 147 | 584 | 6.37 | 0.61 | 0.00 | 0.02 | 0.61 | 0.90 |
| | 3 | 149 | 871 | 6.77 | 0.49 | 0.04 | 0.09 | 0.50 | 0.90 |
| | 12 | 150 | 1300 | 7.17 | 0.28 | 0.03 | 0.01 | 0.28 | 0.90 |
| | 11 | 150 | 4105 | 8.32 | 0.23 | 0.06 | 0.05 | 0.24 | 0.90 |
| | 1 | 150 | 14328 | 9.57 | 0.25 | 000 | 0.03 | 0.25 | 0.90 |
| KREC | 13 | 150 | 1064 | 6.97 | 0.43 | 000 | 0.02 | 0.43 | 1.49 |
| | 12 | 150 | 4403 | 8.39 | 0.24 | 0.03 | 0.03 | 0.25 | 1.49 |
| | 1 | 150 | 4817 | 8.48 | 0.26 | 0.03 | 0.01 | 0.26 | 1.49 |
| | 3 | 150 | 5486 | 8.61 | 0.21 | 0.01 | 0.04 | 0.22 | 1.49 |
| | 11 | 150 | 11968 | 9.39 | 0.23 | 0.06 | 0.03 | 0.24 | 1.49 |
| | 2 | 147 | 22026 | 10.0 | 0.27 | 000 | 0.08 | 0.28 | 1.49 |

Table 26. TREC and KREC precision analysis with between variation estimates across paper manufacturers

| Analyte | Sample | N | Mean copies/10^{6} cells | Within Paper Lot Lognormal CV% | Between Paper Lognormal CV% | Between Manufacturer Lognormal CV% | Total Lognormal CV% |
| --- | --- | --- | --- | --- | --- | --- | --- |
| TREC | 2 | 147 | 584 | 67 | 0 | 6 | 67 |
| | 3 | 149 | 871 | 52 | 4 | 2 | 53 |
| | 12 | 150 | 1300 | 28 | 3 | 9 | 29 |

K203035 - Page 33 of 43

{33}

| Analyte | Sample | N | Mean copies/105cells | Within Paper Lot Lognorma 1 CV% | Between Paper Lognormal CV% | Between Manufacturer Lognormal CV% | Total Lognormal CV% |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | 11 | 150 | 4105 | 24 | 6 | 1 | 25 |
| | 1 | 150 | 14328 | 25 | 0 | 5 | 25 |
| KREC | 13 | 150 | 1064 | 45 | 0 | 3 | 45 |
| | 12 | 150 | 4403 | 25 | 3 | 2 | 25 |
| | 1 | 150 | 4817 | 26 | 3 | 3 | 26 |
| | 3 | 150 | 5486 | 21 | 1 | 1 | 22 |
| | 11 | 150 | 11968 | 23 | 6 | 4 | 24 |
| | 2 | 147 | 22026 | 27 | 0 | 3 | 28 |

ANOVA results in Table 27 below demonstrate that there were statistically significant differences between paper manufacturers (Sample #2/KREC) and between paper lots (Sample #11/TREC and KREC); however, the highest between lot variation was 0.06 (Sample #11/TREC and KREC), and the highest between manufacturer variation was 0.09 (Sample #2/KREC). Per study acceptance criteria, the variation between these samples would not be clinically significant. The data demonstrated the similarity in performance between two major brands of filter paper.

Table 27. TREC and KREC between-paper lot variation and between-paper manufacturer variation, and the corresponding p-values from ANOVA

| Analyte | Sample | N | Mean Copies (copies/105cells) | Mean Ln Copies | Between Paper Lot SD | P-value for testing level differences between paper lots | Between Manufacturer SD | P-value for testing level differences between manufacturers |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TREC | 2 | 147 | 584 | 6.37 | 0.00 | 0.781 | 0.02 | 0.759 |
| | 3 | 149 | 871 | 6.77 | 0.04 | 0.272 | 0.09 | 0.133 |
| | 12 | 150 | 1300 | 7.17 | 0.03 | 0.226 | 0.01 | 0.725 |
| | 11 | 150 | 4105 | 8.32 | 0.06 | 0.011 | 0.05 | 0.091 |
| | 1 | 150 | 14328 | 9.57 | 0.00 | 0.776 | 0.03 | 0.291 |
| KREC | 13 | 150 | 1064 | 6.97 | 0.00 | 0.206 | 0.02 | 0.652 |
| | 12 | 150 | 4403 | 8.39 | 0.03 | 0.736 | 0.03 | 0.365 |
| | 1 | 150 | 4817 | 8.48 | 0.03 | 0.063 | 0.01 | 0.622 |
| | 3 | 150 | 5486 | 8.61 | 0.01 | 0.262 | 0.04 | 0.143 |
| | 11 | 150 | 11968 | 9.39 | 0.06 | 0.009 | 0.03 | 0.291 |
| | 2 | 147 | 22026 | 10.0 | 0.00 | 0.726 | 0.08 | 0.017 |

K203035 - Page 34 of 43

{34}

# C Clinical Studies:

(i) Specimens

A clinical study was conducted at one foreign testing site, the Statens Serum Institute (SSI) in Denmark. SSI is the same site that conducted the study for the PerkinElmer EnLite Neonatal TREC Assay (DEN140010). The majority of the DBS specimens tested for the clinical study (retrospective, anonymized residual [leftover] newborn specimens) were procured by SSI from the screened Danish population. Specimen collection was prospective-retrospective and enriched with confirmed positive specimens for SCID and XLA which came from two biobanks: Danish Neonatal Screening Biobank and the California Biobank. Additionally, 3 of the 4 confirmed positive samples for XLA were procured from the SSI. Confirmed positive specimens were also procured from the California Department of Public Health Genetic Disease Screening Program Biobank (CA), including SCID (all 17 specimens) and XLA (1 of 4 confirmed specimens). All samples were tested at SSI as part of the evaluation. Age matched control specimens were used and presumed to be normal and were archived just before and after the time period during which the confirmed positive specimens for each of the two disorders (SCID, XLA) were archived. These specimens were used as controls for storage conditions to ensure specimen integrity remained intact within the timeframe during which they were stored. Therefore, in addition to consecutive specimens, archived specimens with storage time matched to the storage time of positive specimens were included. Specimen inclusion/exclusion criteria were provided and included specimen age, and storage conditions.

(ii) Inclusion/Exclusion criteria for assessment of specimens

The age of the newborns at the time of specimen collection was less than or equal to 7 days to mimic the expected intended use population and a typical sample collection age for first tier screening to enable early diagnosis. Routine newborn screening specimens originating from the study site were collected at least 5.5 years ago to be able to assess, based on medical records, that the subject had not been identified to present clinical signs attributable to SCID or XLA. The confirmed positive SCID or XLA specimens had been collected ≤ 17 years before the start date of the study to avoid inclusion of sample stored for longer than that time period. Confirmed positive XLA samples were difficult to procure; therefore, samples violating the upper limit of the storage time or the limit for the age of the newborn at the time of sample collection were included.

Inclusion criteria included the following:

- Age of the newborn at the time of specimen collection ≤ 7 days
- Specimen storage conditions are known
- Specimen collected at least 5.5 years ago (from study start)

For specimens confirmed positive for SCID:

- Specimen confirmed positive for typical SCID (documentation of confirmed diagnosis and/or documentation of flow cytometry result)
- Date of specimen collection is ≤ 17 years from the start date of the study

For specimens confirmed positive for XLA:

K203035 - Page 35 of 43

{35}

- Specimen confirmed positive for XLA (e.g., documentation of BTK gene mutation and/or flow cytometry result)
- Date of specimen collection is ≤ 17 years from the start date of the study

In addition, for storage age-matched control specimens:
- Date of specimen collection is known
- Specimen collected just before or after the time period during which the confirmed positive specimens for SCID and/or XLA was collected

Exclusion criteria included the following:
- The DBS is not of appropriate quality (i.e., DBS is scratched, abraded, show signs of incomplete saturation or supersaturation, dilution, discoloration, or contamination)
- Parents/guardians have opted out for use of the specimen beyond routine screening
- Repeat specimen from a newborn whose specimen is already included in the study
- Less than one disk with a diameter of 3.2 mm (1/8 inch) of DBS available in the cut-off phase, and less than two disks available in the pivotal phase for routine specimens

In addition, for specimens confirmed positive for SCID:
- Other than typical SCID, such as less acute SCID (e.g., leaky SCID, variant SCID) or SCID like syndromes (e.g., DiGeorge syndrome or Omenn syndrome)

In addition, for specimens confirmed positive for XLA:
- Atypical XLA or female carriers

(iii) Study Design:

A total of 6924 unique specimens were tested in the study: 3432 specimens were tested in the cut-off phase and 3484 specimens were tested in the pivotal phase (this number includes 17 SCID cases, 6 XLA cases and 52 SMA cases; SMA cases are deducted from the table – refer to DEN200044). The main study consisted of two phases. Specimens in the cut-off study were not retested in the pivotal study.

Cut-off Phase: Newborn specimens representative of samples of the target population at the study site were tested with the device to produce newborn population distribution data for TREC and KREC and to select clinical cut-off values for the pivotal study. The number of false positives for TREC and KREC were determined from the data using lower percentiles 0.3%, 0.5, and 1% and are shown in Table 28.

Table 28. Descriptive statistics for the copies (per 10⁵ cells) for KREC and TREC and the percentile cut-offs used in the study

| Analyte | Copies / 10⁵ cells | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Median | Min | Max | 0.3 percentile | 0.5 percentile | 1.0 percentile |
| TREC | 2520 | 117 | 9990 | 262 | 413 | 563 |
| KREC | 3590 | 12 | 20100 | 129 | 261 | 484 |

K203035 - Page 36 of 43

{36}

Pivotal Phase: Presumed unaffected newborn specimens representing the target population at the study site were supplemented with confirmed positive specimens collected from SCID and 6 XLA affected newborns. A total of 3432 unique samples as shown in Table 29 (after subtracting 52 SMA cases – refer to DEN200044), were assessed in the pivotal phase. After exclusion of the samples not fulfilling the inclusion/exclusion criteria, a total of 3383 samples were included in the final analysis. Demographic characteristics of the routine screening specimens and confirmed positive samples were collected. These included: age at the time of sample collection (mean 2.5 days); weight distribution of the newborns at the time of specimen collection (95% ≥ 2500g); gestational age (98% ≥ 34 weeks); and gender (50.6% male and 49.4% female).

Table 29. Summary of Pivotal Phase Specimens Assayed and Included in the Data Analysis

| Sample | Assayed Samples | Excluded due to failure to meet inclusion/exclusion criteria | Excluded due to Invalid Result | Samples included in final analysis |
| --- | --- | --- | --- | --- |
| Routine screening specimens | 3044 | 26 | - | 3018 |
| SCID positives | 17 | | - | 17 |
| XLA positives | 6 | | - | 6 |
| Storage time matched controls | 365 | 23 | - | 342 |
| Total | 3432 | 49 | 0 | 3383 |

Confirmatory test results were used as the comparator for the confirmed positive SCID or XLA cases including the presence of BTK mutation for XLA. Determination of the true clinical status with regard to SCID has been previously described (DEN140010). The true clinical status with regard to XLA was assessed using data available in patient registries. The clinical assessment of the study subjects was obtained from their medical records to confirm that the subject, at 5.5 years of age (defined as 66 months) or younger, has not been identified with XLA (has no clinical signs attributable to XLA) and is apparently healthy. Danish hospital registry data were reviewed by filtering out subjects that had at least one of the 275 ICD10 diagnosis codes that were listed as potentially linked to XLA. By the age of 5.5 years, a subject with a XLA would have either:

- Been diagnosed with the XLA; or
- Presented with serious medical complications suggestive of XLA as listed below:

Table 30. Symptoms and signs associated with XLA

| Age of onset | Symptoms, signs |
| --- | --- |
| 0-2 years | A severe life-threatening bacterial infection such as sepsis, meningitis, cellulitis, or empyema |

K203035 - Page 37 of 43

{37}

To establish the screening performance, the cut-offs for TREC and KREC were determined by calculating the TREC and KREC concentrations corresponding to the 0.3th and 1.0th population percentiles (262 copies/10 $^5$ cells for TREC, 484 copies/10 $^5$ cells for KREC) established in the cut-off study with an independent dataset and were applied…