(299 days)
Not applicable.
Not Found
No
The summary describes a standard in vitro diagnostic kit and instrument for measuring DNA levels. There is no mention of AI, ML, or any algorithms beyond basic data processing and cut-off determination.
No
The device is an in vitro diagnostic device used for screening newborns for severe combined immunodeficiency disorder (SCID) and does not directly treat or prevent a medical condition.
Yes
The "Intended Use / Indications for Use" section explicitly states, "The EnLite™ Neonatal TREC Kit is an in vitro diagnostic device..."
No
The device description explicitly states that the device is comprised of a kit containing reagents, an instrument (VICTOR™ EnLite), and software. This includes hardware components beyond just software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Explicit Statement: The "Intended Use / Indications for Use" section explicitly states: "The EnLite™ Neonatal TREC Kit is an in vitro diagnostic device..."
- Definition of IVD: An in vitro diagnostic device is a medical device intended for use in vitro for the examination of specimens, including blood and tissue samples, derived from the human body, solely or principally for the purpose of providing information concerning a physiological or pathological state or a congenital abnormality. This device fits this definition perfectly as it analyzes blood specimens to aid in screening for SCID.
- Intended Use: The intended use is to determine TREC DNA in blood specimens to aid in screening newborns for SCID, which is a physiological/pathological state.
- Specimen Type: It uses blood specimens, which are common in vitro diagnostic samples.
N/A
Intended Use / Indications for Use
The EnLite™ Neonatal TREC Kit is an in vitro diagnostic device intended for the semiquantitative 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.
Product codes (comma separated list FDA assigned to the subject device)
PJI, KHO
Device Description
The EnLite™ Neonatal TREC Kit is comprised of the EnLite™ Neonatal TREC Kit, the VICTOR™ EnLite instrument and the EnLite™ workstation software. The EnLite™ Neonatal TREC Kit contains reagents sufficient for 384 reactions or 1152 reactions, and multi-level, dried blood spot (DBS) calibrators and controls. The DBS calibrators and DBS controls have been prepared from porcine whole blood with a hematocrit value of 48-55%, and contain purified salmon-sperm, TREC, and beta-actin DNA.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Blood specimens dried on filter paper (from newborns)
Indicated Patient Age Range
Newborns
Intended User / Care Setting
For prescription use only. Clinical laboratory setting (implied by newborn screening laboratory, external sites, and need for specialized equipment)
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
Cut-off Establishment Study:
- Sample size: 3243 archived, retrospective newborn specimens. After sample elimination due to failure to meet specimen inclusion/exclusion criteria, 2846 samples were included in the analysis.
- Data source: Danish Newborn Screening Biobank (NBS-Biobank). Samples originated from the Danish population, were leftover dried blood spot (DBS) samples collected on devices intended for newborn screening.
- Annotation protocol: The 2.5 percentile was identified as the cut-off for use in the pivotal clinical validation study. The cut-off is 36 copies/uL for TREC and 56 copies/uL for beta-actin.
Pivotal Study:
- Sample size: A total of 6389 Danish newborn screening biobanked newborn routine DBS samples, and 82 enrichment samples (17 confirmed SCID positive samples, 9 confirmed low-level TREC specimens and 56 samples used for blinding purposes) for a total of 6471 neonatal specimens. Of these, 6373 were included in the analysis population after removals for inclusion/exclusion criteria violations and other reasons. For performance measurement, 5454 specimen results (from 5437 routine newborn specimens) along with the 17 confirmed positive SCID cases were used.
- Data source: Archived, retrospective dried blood spot samples submitted to the laboratory for routine newborn screening and stored in the Danish biobank. 17 archived confirmed SCID positive DBS specimens obtained from newborn screening laboratories in the US.
- Annotation protocol: Inclusion criteria for the pivotal study included age of the newborn at time of sample collection 30% drop after 7 days. High temperature (+35 °C) and humidity (RH 80%) can lead to >50% drop in 7 days, and ~80% after 7 days. Recommended long-term storage: plastic bags with desiccant at -30 to -16 ℃.
- Reagent Stability (In Use and On Board):
- Study type: In-use and on-board stability of kit components, including a transport simulation treatment.
- Key results: Elution buffer can be stored at 2-8°C for 14 days dilated. Reagent mixture can be stored at 2-8°C for 23 hours prepared. Calibrators and controls can be thawed/refrozen 4 times, stored -30 to -16°C for 14 days in re-sealable bag with desiccant. Other reagents (concentrate, reaction buffer, DNA Polymerase, PCR diluent) can be thawed/refrozen 4 times, stored -30 to -16°C for 14 days. Microplates with punched samples can be stored at room temp (19-25°C) for 60 minutes after PCR and hybridization before measurement.
- Reagent Stability (In Use and On Board):
- d. Detection limit:
- Study type: Determination of Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ).
- LoB sample size: Five dried blood spot samples, with 10 separate runs of 6 replicates/sample (60 results/sample per DBS).
- LoD/LoQ sample size: Five low-level samples, with 27 runs performed using 5 samples with 4 replicates/sample (108 results per sample).
- Key results: LoB was 3 copies/uL. LoD was 20 copies/uL. LoQ was 29 copies/uL.
- e. Analytical specificity (Interfering Substances, Primer and Probe Specificity, Specimen Carryover Contamination):
- Interfering Substances:
- Study type: Evaluation of potential for interfering endogenous substances (bilirubin, lipids, hemoglobin, heparin).
- Key results: Unconjugated bilirubin, conjugated bilirubin, heparin, and lipemic samples did not interfere. Hemoglobin had an interfering effect; concentrations lower than 17 g/L did not interfere.
- Primer and Probe Specificity:
- Study type: In silico analysis for cross-reactivity.
- Key results: No homology observed leading to false results.
- Specimen Carryover Contamination:
- Study type: Evaluation of analyte carryover by puncher instrument.
- Sample size: N=8 repetitions of the high-low-low cycle for each of three puncher instruments.
- Key results: Carryover study met predefined acceptance criteria. Highest observed mean carry-over percentage was 1.5%.
2. Comparison studies:
- Interfering Substances:
- b. Matrix comparison (Between Filter Paper Lot-to-Lot Reproducibility):
- Study type: Evaluation of manufacturer differences and lot differences for filter paper, homogeneity of analyte across DBS.
- Sample size: 36 total (6 levels x 6 lots). Each sample was analyzed using one kit lot and three different thermocyclers from the same manufacturer.
- Key results: Within the measuring range, within-run variation 29-47%, between-run 28-32%, between paper-lot 0.02-0.05%, between paper-manufacturer 0.01-0.04%, total variation 44-56%. Imprecision was higher outside of the measuring range. No significant differences between paper manufacturers or between paper lots. Homogeneity was equal between different paper manufacturers and lots.
3. Clinical studies:
- a. Establishment of the cut-off:
- See description in "Description of the test set, sample size, data source, and annotation protocol" above.
- b. Pivotal Study:
- See description in "Description of the test set, sample size, data source, and annotation protocol" above.
- Key results:
- Initial testing round: All 17 confirmed SCID specimens identified as below cut-off. An additional 94 routine newborn screening samples were retested, total of 111 retested.
- After second round of testing (final classification): All but one of the 17 confirmed SCID positive specimens classified as presumptive SCID screen positive. One SCID positive specimen was classified as invalid due to beta-actin amplification failure.
- Overall Percent Agreement (OPA) (excluding invalid results): (16 + 5410) / 5442 * 100% = 99.7 % (95% CI from 99.4% to 99.8%)
- Positive Percent Agreement (PPA) (excluding invalid results): (16 / 16) * 100% = 100 % (95% CI from 79.4% to 100%)
- Negative Percent Agreement (NPA) (excluding invalid results): (5410 / 5426) * 100% = 99.7 % (95% CI from 99.4% to 99.8%)
- Retest rate: 1.9%
- False positive rate: 1.5% in the first round of testing; 0.5% after repeat testing on follow-up cases.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
- Positive Percent Agreement (PPA): 100% (95% CI: 79.4% to 100%)
- Negative Percent Agreement (NPA): 99.7% (95% CI: 99.4% to 99.8%)
- Overall Percent Agreement (OPA): 99.7% (95% CI: 99.4% to 99.8%)
- Retest rate: 1.9%
- False positive rate (first round): 1.5%
- False positive rate (final): 0.5% (after repeat testing on follow-up cases)
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.
Not applicable.
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.
0
EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR ENLITE™ NEONATAL TREC KIT DECISION SUMMARY
A. DEN Number:
B. Purpose for Submission:
De novo request for evaluation of automatic class III designation of the EnLite™ Neonatal TREC Kit
C. Measurand:
T-Cell Receptor Excision Circles (TRECs)
D. Type of Test:
Semi-quantitative, polymerase chain reaction (PCR) based nucleic acid amplification and timeresolved fluorescence resonance energy transfer (TR-FRET) based detection
E. Applicant:
Wallac Oy, a PerkinElmer subsidiary
F. Proprietary and Established Names:
EnLite™ Neonatal TREC Kit
G. Regulatory Information:
-
- Regulation section:
21CFR 866.5930
- Regulation section:
-
- Classification:
Class II
- Classification:
-
- Product code:
- PJI Severe Combined Immunodeficiency Disorder (SCID) Newborn Screening Test System
- KHO Fluorometer, For Clinical Use
1
4. Panel:
Immunology
H. Intended Use:
-
- Intended use:
The EnLite™ Neonatal TREC Kit is an in vitro diagnostic device intended for the semiquantitative 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).
- Intended use:
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.
-
- Indication(s) for use:
Same as intended use above.
- Indication(s) for use:
-
- Special conditions for use statement(s):
For prescription use only.
- Special conditions for use statement(s):
-
- Special instrument requirements:
VICTOR™ EnLite instrument and the EnLite™ workstation software.
- Special instrument requirements:
Thermal cycler compatible with full-skirted 96-well PCR plates and with heated lid, heating rate of 3°C - 6°C /sec, cooling rate of 3°C - 4.5°C /sec, heated lid setting using 100°C - 105°C or tracking mode of 5℃ above the block temperature.
I. Device Description:
The EnLite™ Neonatal TREC Kit is comprised of the EnLite™ Neonatal TREC Kit, the VICTOR™ EnLite instrument and the EnLite™ workstation software. The EnLite™ Neonatal TREC Kit contains reagents sufficient for 384 reactions or 1152 reactions, and multi-level, dried blood spot (DBS) calibrators and controls. The DBS calibrators and DBS controls have been prepared from porcine whole blood with a hematocrit value of 48-55%, and contain purified salmon-sperm, TREC, and beta-actin DNA. Specific contents of the kit are as follows:
2
| DBS calibrators | Filter paper cassettes containing dried blood spots with
three levels (A, B, C) of TREC and beta-actin. The
exact TREC and beta-actin concentrations are given on
the lot-specific quality control certificate included in
the kit but approximate values are targeted as follows:
A: TREC 25 copies/µL and beta-actin 18 copies/ µL
B: TREC 240 copies/µL and beta-actin 120 copies/ µL
C: TREC 770 copies/µL and beta-actin 370 copies/ µL |
|------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| DBS controls | Filter paper cassettes containing dried blood spots with
one negative control and two positive controls (C1, C2,
C3) for TREC and two levels of beta-actin. The values
for the DBS controls measured by the manufacturer are
given on the lot specific quality control certificate
included in the kit. Each laboratory should establish its
own mean and acceptable range but approximate
values are targeted as follows:
C1 Low Control: TREC 120 copies/µL and beta-actin
20 copies/ µL
C2 No TREC control: TREC 0 copies/µL and beta-
actin 600 copies/ µL
C3 High Control: TREC 670 copies/µL and beta-actin
600 copies/ µL |
| Elution Diluent | PCR-grade water |
| Reagent Concentrate | Ready-for-use Tris-HCl buffered (pH 8) salt solution
with EDTA, Tween® 20, deoxyribonucleotide
triphosphates, unlabeled oligonucleotides, labeled
oligonucleotides, and bovine serum albumin. |
| 5x Reaction Buffer | Ready-for-use buffer with 7.5 mM MgCl2 |
| DNA Polymerase | Ready-for-use solution with 20 mM Tris-HCl (pH 7.4
at +25 °C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl,
stabilizers, 200 µg/mL bovine serum albumin, and 50
% glycerol |
| PCR Diluent | Ready-for-use water solution with MgCl2 |
| Barcode labels for the plate | |
| Lot-specific quality control certificate | |
3
J. Substantial Equivalence Information:
-
- Predicate device name(s) and DEN number(s):
Not applicable.
- Predicate device name(s) and DEN number(s):
-
- Comparison with predicate:
Not applicable.
- Comparison with predicate:
K. Standard/Guidance Document Referenced:
CLSI NBS01-A6: Blood Collection on Filter Paper for Newborn Screening Programs; Approved Standard-Sixth Edition
L. Test Principle:
The EnLite™ Neonatal TREC Kit is a combination of PCR-based nucleic acid amplification and time-resolved fluorescence resonance energy transfer (TR-FRET) based detection. The test detects two targets: TREC (the marker for SCID) and beta-actin (an internal control for the test). Amplification and detection of Beta-actin is used to monitor the PCR amplification of a specimen to aid in detecting samples that do not have a sufficient amount of DNA for analysis. Beta-actin is used as a control for monitoring specimen amplification. The determination of TREC and beta-actin is performed simultaneously for each specimen.
The EnLite™ Neonatal TREC Kit is designed to measure TREC and beta-actin DNA in newborn dried blood spots from filter paper and involves the following steps: (1) A small punch is made from a dried blood spot (DBS) specimen from a filter paper intended for newborn screening; (2) The DBS punch, and reagents are added to a 96 well microtiter plate; calibrators and controls are also added to separate wells in the plate (3) TREC DNA is eluted during incubation in a thermocycler; (3) Target sequence-specific TR-FRET probes, which include donor and acceptor fluorophores coupled to opposite ends of a single probe molecule, are added to the microtiter plate; (4) The microtiter plate is returned to the thermocycler for DNA amplification and hybridization. Upon hybridization with the target sequence, energy transfer towards the acceptor emits a specific fluorescence at a given wavelength based on prolonged decay time of the energy-transfer induced acceptor signal; (5) Signal detection is performed on the Victor™ EnLite Instrument bench top microtiter plate fluorometer. The instrument automatically measures the microtiter plates and outputs quantitative results based on the measured fluorescence signals.
The EnLite™ workstation software interprets test results utilizing fluorescence counts measured at 615 nm, 665 nm, and 780 nm. Corrected fluorescence counts, "TREC Response" and "Betaactin Response" for all reactions are calculated from the raw fluorescence counts. The result interpretation is based on two separate calibration curves which use blanks and DBS calibrators A-C. The response, corrected counts, are fitted against the ArcSinh transformed concentrations (copies/uL) using the unweighted linear regression. The test quality control is based on three kit control result interpretations. Each test generates results for TREC and beta-actin.
4
M. Performance Characteristics:
1. Analytical performance:
a. Reproducibility/Precision:
Three reproducibility studies were conducted using the EnLite™ Neonatal TREC Kit. These included a site-to-site reproducibility study, an independent precision study to evaluate the within-lot, between-lot and total variation of the EnLite™ Neonatal TREC Kit and a thermocycler reproducibility study.
Study I. Site-to-Site reproducibility:
The reproducibility of the EnLite™ Neonatal TREC Kit was assessed at three clinical sites: two external sites in the US and one non-US internal site. The reproducibility study at each clinical site was conducted over 5 non-consecutive days, using one lot with two operators per site performing 5 runs each (10 runs per laboratory). A run consisted of one plate with 3 replicates per sample. The total number of measurements was 90 per sample (10 runs x 3 laboratories x 3 replicates/sample). Each run included calibrators in triplicate and controls.
The reportable range of the test is 29-473 TREC copies/uL. Results below 29 are reported as 473. However for the purpose of demonstrating reproducibility of the test, six levels of TREC specimens ranging from 3 copies/uL to 550 copies/uL were tested by all three sites. Four specimens (TR2-TR5) were within the measuring range of the test (29-473 TREC copies/uL) (4)
The lowest
TREC level (TR1), below the measuring range, was prepared from adult blood only containing low to zero TREC. The highest TREC level specimen (TR6) above the measuring range was prepared by spiking adult blood with TREC plasmid DNA. b(4)
Samples were then processed in accordance with the instructions for use (package insert).
Table 1 summarizes the within-laboratory, between-laboratory, within-run, between-run and total variation of TREC results across three clinical sites. These results demonstrate that within the measuring range of the test (29-473 TREC copies/ uL), the within-laboratory percent coefficient of variation (%CV) of the EnLite™ Neonatal TREC Kit was 41.7 to 52.1%, the between-laboratory variation range was 15.1-24.3%, the within-run variation was 31.8 to 47.4%, the between-run variation was 12.0 to 36.1% and the total variation of the test range was 48.5 to 59.4%. Imprecision was larger when specimens had TREC values outside the measuring range. Table 2 summarizes the variation for the within-laboratory, betweenlaboratory, within-run, between-run and total variation for the controls.
Table 1. Within-laboratory, between-laboratory, within-run, between-run and total
5
| Sample | n | Mean TREC
copies /µl | Within
laboratory
variation
% CV | Between
laboratory
variation
% CV | Within
Run
Variation
%CV | Between
Run
Variation
%CV | Total
variation
% CV |
|--------|----|-------------------------|-------------------------------------------|--------------------------------------------|-----------------------------------|------------------------------------|----------------------------|
| 1 TR1 | 90 | 3.16 | 119.1 | 20.2 | 110 | 29.6 | 123.0 |
| e TR2 | 90 | 56.26 | 46.2 | 15.1 | 39.4 | 21.2 | 48.5 |
| TR3 | 90 | 68.03 | 48.5 | 21.2 | 47.4 | 12.0 | 54.5 |
| 2 TR4 | 90 | 200.34 | 41.7 | 22.3 | 31.8 | 26.4 | 48.5 |
| TR5 | 90 | 287.15 | 52.1 | 24.3 | 35.0 | 36.1 | 59.4 |
| TR6 | 90 | 544.57 | 69.8 | 41.7 | 46.2 | 47.4 | 86.9 |
variation of TREC results across three clinical sites
Table 2. Within-laboratory, between-laboratory, within-run, between-run and total variation for the three test controls.
| Sample | n | Mean
TREC
copies
/µl | Within
laboratory
variation
% CV | Between
laboratory
variation
% CV | Within
run variation
%CV | Between
run variation
%CV | Total
variation
% CV |
|-----------------|----|-------------------------------|-------------------------------------------|--------------------------------------------|--------------------------------|---------------------------------|----------------------------|
| No TREC control | 60 | 1.86 | 88.4 | 13.1 | 62.0 | 54.5 | 91.5 |
| Low control | 60 | 63.43 | 88.4 | 39.4 | 86.9 | 15.1 | 102.9 |
| High control | 60 | 262.43 | 104.6 | 30.7 | 94.7 | 35.0 | 113.5 |
Study 2. Precision: Within run, Within-Lot, Between-lot and Between Instrument Reproducibility
A study was conducted to determine the precision of the EnLite™ Neonatal TREC Kit among multiple operators, days, lots, instruments, and thermocyclers. The study consisted of 3 kit lots, 3 Victor™ EnLite instruments and 3 thermocyclers in various combinations (4 replicates per sample x 3 lots x 3 instruments = 36 results). One of five different operators was randomized to perform each run. Twenty-seven (27) runs were performed over 20 days. Ten TREC samples spanning the range of TREC values 3-473 copies/uL were assessed. For evaluation of TREC precision, 8 samples were prepared from adult EDTA whole blood spiked with TREC plasmid DNA; one low level TREC sample was prepared from un-spiked adult blood, and one sample consisted of cord blood (containing endogenous TREC). For evaluation of beta-actin precision, 7 beta-actin samples from porcine whole blood were chosen to span the range of 16-608 copies/uL beta-actin. For the beta-actin samples, the blood was spiked with TREC plasmid DNA to obtain constant TREC values in all samples.
Results for the TREC precision within-run, within-lot, between-run, between-run, betweeninstrument and total variation for TREC samples 2 through 10 are as follows: Within the measuring range of the test, %CV for within-run ranged from 45% to 88%, the % CV for between-run ranged from 18 to 42%, % CV for within-lot ranged from 67% to 101%, and % CV for between-lot ranged from 11% to 33%, between-instrument variation ranged from 0 to 50% and total %CV ranged from 73% to 106%. Outside of the measuring range, imprecision was greater. Sample 1 had mean TREC copies/uL of 2 b(4)
6
Testing of sample 4 yielded one outlier.
A summary of the variation (%CV) for the within-run, between-run, within-lot, between-lot, between instrument and total variation components for beta-actin are as follows: Within the measuring range, the %CV for the precision results for beta-actin within-run were 46-71%, the %CV range for between-run was 7-37%. the %CV range for within-lot was 55-95%, the %CV range for between-lot was 10-24%, the %CV range for between-instrument was 0 to 21% and the range of variation for total beta-actin the %CV range was 56-100%.
The distribution of results of the reproducibility study categorized by the cut-off (36 copies/uL) is shown in Table 3. Results below the cut-off would be deemed SCID positive, while results above the cut-off would be deemed SCID negative.
| Sample | N | Mean TREC
copies /µl | Below Cut-off
(%) | Above Cut-off
(%) |
|--------|-----|-------------------------|----------------------|----------------------|
| 1 | 108 | 3 | 108 (100%) | |
| 2 | 108 | 19 | 83 (77%) | 25 (23%) |
| 3 | 108 | 29 | 70 (65%) | 38 (35%) |
| 4 | 108 | 43 | 41 (38%) | 67 (62%) |
| 5 | 108 | 59 | 25 (23%) | 83 (77%) |
| 6 | 108 | 74 | 18 (17%) | 107 (100%) |
| 7 | 108 | 144 | | 108 (100%) |
| 8 | 108 | 276 | | 108 (100%) |
| 9 | 108 | 473 | | 108 (100%) |
| 10 | 108 | 1790 | | 108 (100%) |
Table 3: Dichotomized Results for Precision Samples
Study 3. Thermocycler Reproducibility
A thermocycler reproducibility study was performed using 3 instruments from different manufacturers testing a panel of samples with varying levels of TREC copies. b(4)
Blood was dispensed onto filter paper from two different paper manufacturers and 12 samples were analyzed in the study b(4)
The study acceptance criteria
was based on between-thermal cycler comparison for 5 levels of TREC copies within the measuring range, which is evaluated by testing for statistically significant differences between thermocyclers. Test calibrators and controls were included in each run. Table 4 shows the within-run, between-run, between paper manufacturer, between-thermal cycler and total variation. Total variation is variation that has been pooled across two distinct paper manufacturers and across all three thermal cyclers. One well of the run performed on the 2nd day of the study showed no signals for TREC or beta-actin in one thermocycler. Investigation revealed that part of the contents of the well was missing due to incomplete seal of the plate
7
and this result was excluded from data analysis. Recommendations for specific thermocycler specifications are made in the instructions for use. The total variation within the measuring range of the test is similar (63-74% CV) between samples. Imprecision is higher outside of the measuring range. Analysis using one-way ANOVA showed that there were no differences between the thermocyclers from the different manufacturers at any of the TREC sample levels.
| Sampl
e | N | Mean
TREC
(copies
/µl) | Within-
run
variation
% CV
(copies/µl) | Between-
run
variation
SD
(copies/µl) | Between-
paper
manufacture
r %CV
(copies/µl) | Between
thermal-
cycler
variation
%CV
(copies/µl) | Total
variation
%CV
(copies/µl) |
|------------|----|---------------------------------|----------------------------------------------------|---------------------------------------------------|----------------------------------------------------------|------------------------------------------------------------------|------------------------------------------|
| TR1 | 60 | 4 | 123 | 0.3 | 5.58 | 0.04 | 123 |
| TR2 | 60 | 52 | 73 | 0.01 | 12.4 | 0.01 | 74 |
| TR3 | 60 | 83 | 51 | 33.6 | 0.01 | 0.01 | 63 |
| TR4 | 60 | 124 | 64 | 13.3 | 0.1 | 0.03 | 66 |
| TR5 | 60 | 194 | 61 | 24.9 | 0.03 | 0.02 | 67 |
| TR6 | 59 | 393 | 59 | 39.9 | 0.07 | 0.01 | 75 |
Table 4. TREC within-run, between-run, between-paper manufacturer, between-thermal cycler and total variation
b. Linearity/test reportable range:
The objective of the study was to evaluate the TREC linearity of the EnLite™ Neonatal TREC Kit. Human adult EDTA whole blood sample was used to represent the low concentration TREC sample. A high concentration samples was prepared by spiking TREC plasmid DNA into the same adult blood specimen. b(4)
Samples with intermediate TREC levels were prepared by pooling the low concentration sample with high concentration sample. A total of 15 samples were prepared, 8 of the samples (sample number 4 through sample number 12) spanned the measuring range of the test (29-473 TREC copies/uL). The dried blood spot (DBS) samples were prepared by spotting the samples using filter paper intended for neonatal screening, b(4)
The TREC levels of the sample blood spots were measured with one kit lot using one thermal cycler and one VICTOR EnLite™ instrument. The different dilutions were scattered in random order and each sample was analyzed in in 5 replicates in a single run. Two separate runs were conducted in the experiment using the same set of samples. To investigate potential outliers of the first run, a second run was conducted. Test calibrators [blank reaction and three DBS calibrators in 3 replicates and Test controls (duplicates)] were included in the run. b(4)
Linearity analysis within the entire range tested was performed using the combined data (runl + run 2). A polynomial evaluation of linearity was used for the data analysis. [4 ]
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All sample dilutions met the pre-defined criteria for acceptance. The EnLite™ Neonatal TREC Kit is linear within the range of 22-1164 copies/uL. The reportable range of the test is 29-473 Copies/uL for TREC and 16 Copies/uL to 608 Copies/uL for beta actin.
Calibrator Linearity
b(4)
An unweighted linear regression algorithm is used for the calibration curve. b(4)
An example calibration graph for TREC from a reproducibility study run is shown in Figure 1 below.
Image /page/8/Figure/4 description: The image shows a scatter plot of TREC copies per microliter. The x-axis represents TREC copies per microliter, ranging from 0 to 5000. The y-axis represents TREC, ranging from 5,000 to 25,000. There is a linear trendline going through the data points.
Figure 1. Calibration graph for TREC from a reproducibility study run
- c. Traceability, Stability, Expected values (controls, calibrators, or methods)
- i. Traceability
9
There is no recognized standard or reference material for TREC DNA. The EnLite Neonatal TREC Kit contains calibrators and controls employed in test calibration and quality control. Several in-house reference materials b(4)
b(4) ) are used in the calibration process and in the value assignment of the test calibrators.
The EnLite™ Neonatal TREC Kit contains calibrators and controls that are provided on a filter paper cassette and are prepared from porcine whole blood dispensed onto filter paper intended for neonatal screening. The blood used to prepare the calibrators and controls is spiked with plasmid DNA containing the TREC target sequence to cover the range of 0-576 copies/uL blood.
Controls
The test consists of the following set of controls that are used to monitor test performance: Blank reactions serve as no template control reactions and there are 3 control DBS specimens, including low control (C1: low TREC, low beta-actin), no TREC control (C2: no TREC, normal beta-actin) and high control (C3: normal TREC, normal beta-actin).
| Control | TREC | Beta-actin |
|---|---|---|
| C1 Low control | 120 | 20 |
| C2 No TREC control | 0 | 600 |
| C3 High control | 670 | 600 |
Calibrators
The EnLite™ Neonatal TREC calibrators consist of blank reactions and three DBS calibrators, A-C, with known quantities of TREC and beta-actin DNA.
| Calibrator | TREC | Beta-Actin |
|---|---|---|
| A | 25 | 18 |
| B | 240 | 120 |
| C | 770 | 370 |
ii. Stability:
Specimen Stability
The objective of the sample stability testing was to define the short term stability (1-28 days) of TREC in dried blood spot samples collected using filter paper based sample collection devices. The study was conducted using various storage conditions. Adult whole blood and cord blood were mixed in various ratios to obtain samples within the desired TREC levels, such that the samples (n=4) would cover the expected measuring range of the EnLite™ Neonatal TREC Kit.
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The test blood samples were spotted using two filter paper brands intended for newborn screening 55 µL/spot and dried overnight at room temperature. Analysis of sample stability was performed using 5 different timepoints: 0, 7, 14, 21 and 28 days. Analysis of the zero timepoint was performed immediately after the drying step using 4 plates containing 5 replicates of the sample stability study samples on each plate. Analysis of the remaining 4 timepoints used 4 plates/timepoint (2 replicates of the sample stability study samples on each plate). Each of these sample plates also included calibrators in triplicate and controls in duplicate.
TREC concentration was measured at the following conditions: storage at -20℃ with no packing, storage at -20°C in a sealed bag with desiccant, storage at 4°C with no packing, storage at 4°C in a sealed bag with desiccant, storage at 22°C, room humidity (RH) at 22-33% with no packing and storage at 35℃, RH 80% with no packing. b(4)
. Figures below shows TREC concentrations measured at different storage conditions and timepoints using 3 kit samples, including 5 TREC copies/uL (Figure 2), 56 TREC copies/uL (Figure 3 and 5) and 273 TREC copies/uL (Figure 4).
As shown in these figures, the decline in the measured TREC concentration was the steepest for samples stored at 35℃, RH 80% with no packing; this was the least optimal storage condition. The decline in the measured TREC concentration was the smallest for samples stored at -20°C. b(4)
Storage of specimens in an environment with elevated temperatures and humidity increases the risk of false positive TREC screening results. TREC concentration may drop over 30% after 7 days at room temperature with low humidity (RH 22-33%). When stored at high temperature (+35 °C) and humidity (RH 80%), the concentration may drop over 50% during the first 7 days, and after 7 days approximately 80%. The results are shown in Figures 2, 3, 4, and 5 for three values of TREC and two filter papers. For longterm storage (up to 19 months), specimens should be placed into plastic bags together with a desiccant and stored at -30 to -16 ℃. The recommended storage condition in the package insert for storage is -30°C to -16°C.
Figure 2. Sample 1 (5 TREC copies/uL) measured at different storage conditions and timepoints on Filter paper brand 1.
11
Image /page/11/Figure/0 description: This figure shows a line graph titled "Sample 1, Filter paper brand 1 (TREC 5 copies / μl)". The x-axis is labeled "Storage time (days)" and ranges from 0 to 30. The y-axis is labeled "TREC copies / μl" and ranges from 0 to 12. There are five lines on the graph, each representing a different storage condition: -20 C in a sealed bag with a desiccant, +4 C no packing, +4 C in a sealed bag with a desiccant, from +19 to +25 C RH 22-33% no packing, and +35 C RH 80% no packing.
Figure 3. Sample 2 (56 TREC copies/uL) measured at different storage conditions and timepoints on Filter paper brand 1.
Image /page/11/Figure/2 description: The image is a line graph titled "Sample 2, Filter paper brand 1 (TREC 56 copies / μl)". The x-axis is labeled "Storage time (days)" and ranges from 0 to 30. The y-axis is labeled "TREC copies / μl" and ranges from 0 to 60. There are five lines on the graph, each representing a different storage condition: -20 C in a sealed bag with a desiccant, +4 C no packing, +4 C in a sealed bag with a desiccant, from +19 to +25 C RH 22-33% no packing, and +35 C RH 80% no packing.
Figure 4. Sample 3 (273 TREC copies/uL) measured at different storage conditions and timepoints on Filter paper brand 1
12
Image /page/12/Figure/0 description: The image is a line graph that shows the stability of TREC copies over time under different storage conditions. The x-axis represents storage time in days, ranging from 0 to 30, while the y-axis represents TREC copies per microliter, ranging from 0 to 300. There are five different storage conditions plotted on the graph, including -20 degrees Celsius in a sealed bag with a desiccant, +4 degrees Celsius with no packing, +4 degrees Celsius in a sealed bag with a desiccant, +19 to +25 degrees Celsius with 22-33% relative humidity and no packing, and +35 degrees Celsius with 80% relative humidity and no packing. The graph shows that the number of TREC copies decreases over time for all storage conditions, but the rate of decrease varies depending on the condition.
Image /page/12/Figure/1 description: The image is a figure with a text description. The text says, "Figure 5. Sample 1 (55 TREC copies/uL) measured at different storage conditions and timepoints on Filter paper brand 2." The figure is about measuring TREC copies under different conditions. The sample has 55 TREC copies per microliter.
Image /page/12/Figure/2 description: The image is a line graph that shows the number of TREC copies per microliter over a period of 30 days. There are five lines on the graph, each representing a different storage condition. The storage conditions are -20 degrees Celsius in a sealed bag with a desiccant, +4 degrees Celsius with no packing, +4 degrees Celsius in a sealed bag with a desiccant, from +19 to +25 degrees Celsius with RH 22-33% with no packing, and +35 degrees Celsius with RH 80% with no packing. The graph shows that the number of TREC copies decreases over time for all storage conditions, but the rate of decrease varies depending on the storage condition.
Reagent Stability
The objective of this study was to demonstrate the in-use and on-board stability of the EnLite Neonatal TREC Kit components. These studies were performed with or without an additional transport simulation treatment that simulates the worst case conditions that can occur during packaging and shipping.
In Use Reagent Stability Study
For the in use reagent study, elution diluent and reaction buffer were thawed, diluted as appropriate and stored at 2 to 8° for 15 days before use. All other kit components were thawed and frozen for 4 cycles over 4 days (stored at room temperature for one
13
hour per day and then re-frozen). Calibrators and control cassettes were also opened during thawing and then packed in a re-sealable bag with desiccant. The kit components were otherwise stored at their normal storage conditions (-30 to -16℃). Fourteen days from the first freeze-thaw cycle, reagents were thawed for a fifth time and tested as follows. Calibrators, controls and DBS samples were punched into microtiter plates, which were stored at room temperature (19 to 25°C) and tested after 23 hours. Before starting the test, the reagent mixture was prepared and the mixture was stored at 2 to 8° for 24 hours.
On Board Study
For the on board study, the test plates from study 1 were removed from the VICTOR EnLite™ instrument after the first measurement. These test plates were then stored in the thermal cycler (23°C) for 70 minutes and the plates were then measured again with the VICTOR EnLite™ instrument.
Transport Simulation
A transport simulation treatment was performed for all the kit components, and exposed to the transport simulation treatment before the study. The stress condition sequence for the kit components (packed and shipped with blue ice) was: 1 hour at 25°C, 6 hours at -20°C, 6 days at 4°C, 1 day at -20°C, 2 days at 4°C and ≥ 1 day at -20°C. After the transport simulation, all reagents were stored at their normal storage conditions (-30 to -16℃) until starting the in-use study.
The acceptance criteria for the stability tests are based on product performance specifications for precision. Test-specific acceptance limits corresponding to a=0.5% significance level (2SD) and a=0.3% significance level (3SD) are statistically calculated from the precision requirements (population SD). In addition there are acceptance limits for within and between plates variations to assure that all the individual test results contributing to the mean are acceptable.
The in-use and on-board stability testing was performed in a manner such that the EnLite™ Neonatal TREC Kits were first performed with the in-use treated kit components and the plates were measured without delay. The same plates were re-measured when 70 minutes had elapsed from the end of the PCR program. Table 5 below summarizes the mean TREC results of the in-use study (before on-board testing) followed by results measured 70 minutes after completion of the PCR program (after on-board testing). Table 6 below summarizes the mean beta-actin results on the on-board testing study.
These studies demonstrate that the elution buffer can be stored at 2 to 8° for 14 days, once it is diluted, and used in the EnLite™ Neonatal TREC Kit. The reagent mixture can be stored at 2 to 8°C for 23 hours, once it is prepared. For the calibrators and controls, once they are opened, they can be thawed and re-frozen four times, stored at -30 to -16°C for 14 days in a resealable plastic bag with desiccant (protected from light) and used in the EnLite™ Neonatal TREC Kit. The reagent concentrate, reaction buffer, DNA Polymerase and PCR diluent can
14
also be thawed and re-frozen four times, stored at -30 to -16℃ for 14 days and then used in the EnLite™ Neonatal TREC Kit. Microplates with punched calibrators and controls in wells can be stored at room temperature (19 to 25°C) after PCR and hybridization for 60 minutes before measurement.
| Control | Target
(Copies/µL) | Mean TREC concentration
(Copies/µL) | |
|------------------------------|-----------------------|----------------------------------------|---------------------|
| | | Before on-board testing | After on-board time |
| Kit control 1 | 117 | 85 | 86 |
| Kit control 2 | 0 | 0 | 0 |
| Kit control 3 | 470 | 343 | 344 |
| TREC Reference
Control K1 | 67 | 46 | 51 |
| TREC Reference
Control K2 | 118 | 72 | 77 |
| TREC Reference
Control K3 | 287 | 104 | 120 |
Table 5. Mean TREC on-board stability test results
Table 6. Mean beta-actin on-board testing results
| Control | Target
(Copies/µL) | Mean beta-actin concentration
(Copies/µL) | |
|------------------------------------|-----------------------|----------------------------------------------|---------------------|
| | | Before on-board testing | After on-board time |
| Kit control 1 | 52 | 46 | 38 |
| Beta-actin Reference
Control K1 | 56 | 52 | 49 |
| Beta-actin Reference
Control K2 | 80 | 71 | 66 |
d. Detection limit:
The objective of the study was to determine the TREC limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ) for the EnLite™ Neonatal TREC Kit.
Limit of Blank (LoB)
Five dried blood spot samples prepared from porcine whole blood with no endogenous TREC and low basal level of beta actin were used to assess the LoB. b(4)
The blood was dispensed on filter paper intended for neonatal screening b(4) and dried. Repeated measurements of the blank samples were tested against a full calibration curve (blank reaction and three DBS calibrators in 3 replicates). Three kit controls in duplicate were included in each plate. Ten separate runs of six replicates/sample were performed over five operating days (10 runs x 6 replicates = 60 results/sample). Two kit lots and two thermal cyclers/fluorometer instruments were used for the analysis. b(4)
15
b(4) The observed LoB of TREC was determined to be 3 copies/uL, which met the pre-defined acceptance criteria for the EnLite™ Neonatal TREC Kit.
Limit of Detection (LoD) and Limit of Quantification (LoQ)
Five low-level samples (19, 29, 42, 59 and 74 TREC copies/uL) were used for defining the LoD and the LoQ for TREC. The samples were dried blood spot samples prepared from adult EDTA whole blood that contains a low endogenous level of TREC. b(4)
The blood was spiked with TREC plasmid DNA to obtain low TREC samples. As whole blood contains a considerable level of endogenous beta actin, the betaactin level is constant in all samples. The DBS samples were prepared by spotting the blood with different TREC concentration levels on filter paper intended for newborn screening, b(4)
Repeated measurements were carried out using five low level TREC dried blood spot samples. For the study, 27 runs were performed using 5 samples with 4 replicates/sample (27 runs x 4 replicates = 108 results per sample) over 20 operating days. Three kit lots and three thermal cyclers/fluorometer instruments were used for the analysis. Three kit controls in four replicates were included in each plate. LoD, was defined as the 95th percentile of measurements that exceeds the LoB. The LoO is defined as the lowest concentration of the analyte that can be measured with acceptable total variation.
An estimate of the standard deviation for LoD was derived from five low level sample result distributions. b(4)
The most conservative highest kit lot SD was used and the LoD was reported as 20 copies/ uL, b(4) b(4)
Since there is no reference method for the value assignment of TREC concentration for the LoQ study and no international reference standard, a functional sensitivity study was used to define LoQ. The total SD less than or equal to the specification for total variation was fulfilled for samples with TREC concentrations equal or higher than 29 copies/uL. The observed LoQ of 29 copies/uL met the acceptance criteria.
The reported values for TREC are LoB 3 copies/uL; LoD 20 copies/uL and LoQ 29 copies/uL.
- e. Analytical specificity:
Interfering Substances:
The objective of this study was to evaluate the potential for interfering substances
16
(endogenous) in dried blood spot samples on the measurement of TREC concentrations with the EnLite™ Neonatal TREC Kit. The effect of bilirubin (unconjugated 100mg/L blood) and conjugated (166mg/L blood), lipids (15mg/mL blood), hemoglobin (200g/L blood (this hemoglobin concentration exceeded the recommended interferent test concentration of CLSI EP7-A2), and heparin (0.375 mg/mL blood) on test performance was evaluated. Additionally, because preliminary studies suggested that there was interference by hemoglobin, blood pools with three different TREC concentration levels (unspiked blood and two spiked TREC levels) and intermediate hemoglobin levels (155, 170 and 185 g/L blood = 25% pool, 50% pool and 75% pool, respectively) were also prepared for testing. Interference on TREC was tested using three different TREC levels (low, mid, high) using one kit lot. Dried blood spot samples were prepared by dispensing the prepared samples onto filter paper intended for newborn screening, D(4)
Potential interference was measured using the paired-difference method in which the tested substance is added to the sample. For the study acceptance criteria, a bias of greater than 1SD (calculated from the total variation of the test) at the 5% level of statistical significance (α=0.05) is considered analytically significant.
Unconjugated bilirubin at a concentration of 100 mg/mL blood, conjugated bilirubin at a concentration of 166 mg/L blood, heparin at a concentration of 0.375 mg/mL blood, lipemic samples at lipid concentration of 15 mg/l blood did not interfere with the measurement of TREC using the EnLite™ Neonatal TREC Kit. However, hemoglobin did have an interfering effect and dose-response studies were performed. Only the lowest tested hemoglobin level (25% pool) displayed no interference at any of the tested TREC levels. Since the control pool had 138g/L of hemoglobin, and the 25% pool had 155g/L of hemoglobin, the amount of hemoglobin added to the 25% pool was 17 g/L. Hemoglobin at concentrations lower than 17 g/L did not interfere with TREC measurements.
Primer and Probe Specificity
TREC DNA is formed from sequences from chromosome 14. Beta-actin gene is located in chromosome 7. In silico analysis was conducted to assess for the possibility of crossreactivity to other sequences. No homology was observed leading to false results.
Specimen Carryover Contamination:
The objective of this study was to evaluate whether analyte can be carried over from one sample reaction to subsequent sample reactions by the puncher instrument used to punch out blood disks from the DBS sample for analysis. The test was performed using one kit lot of the EnLite™ Neonatal TREC Kit and three Wallac DBS puncher instruments used to punch 1.5 mm blood spot disks for the analysis. There kit controls in duplicate were included in each plate. The high TREC sample to be used in the study was kit calibrator C prepared from b(4) The mid TREC sample to be used was cord blood prepared by spotting onto filter paper b(4) . The zero TREC level was prepared from porcine whole blood, which is not spiked with TREC. b(4)
17
b(4) The carry-over result for each of the three puncher instruments is summarized in the Table 7 below. The carry-over study meets the predefined acceptance criteria for TREC. b(4)
The highest observed
mean carry-over percentage was 1.5%.
Table 7. Mean carry-over percentages separately for each of three Puncher instruments. N=number of repetitions of the high- low-low cycle.
| Puncher | High sample material | Mean carry-over-percentage (%) (N=8) |
|---|---|---|
| 1 | Calibrator C | 0.6 |
| 1 | Cord blood sample | 0.4 |
| 1 | Neonatal DBS sample | 3.7 |
| 2 | Calibrator C | 0.1 |
| 2 | Cord blood sample | 0.0 |
| 2 | Neonatal DBS sample | 0.6 |
| 3 | Calibrator C | -0.1 |
| 3 | Cord blood sample | 0.1 |
| 3 | Neonatal DBS sample | 0.1 |
f Assay cut-off:
The test reportable range for TREC is 29 copies/uL to 473 copies/uL blood. Samples that result in values below 29 copies/uL blood are reported as 473 copies/uL blood. The reportable range for beta-actin is demonstrated to be from 16 copies/ uL blood to 608 copies/ uL blood. The clinical cut-offs are as follows:
TREC positive: ≥36 copies/uL TREC negative: 473 | 26 | 32 | 39 | 41 | 52 |
Table 12. Distribution of Beta-actin Results
| Population | Nobs | Median | Min | Max | 0.5% | 1.0% | 2.0% | 2.5% | 5.0% |
|---|---|---|---|---|---|---|---|---|---|
| SCID+low | |||||||||
| TREC | 26 | 1028 | 77 | 19900 | NA | NA | NA | NA | NA |
| All Routine | |||||||||
| Samples | 6347 | 691 | 2 | >608 | 17 | 26 | 42 | 48 | 69 |
Of the available 6347 routine newborn specimens, 910 (14%) cases were loss-to-follow due to the absence of proof-of-life documentation at 12 months of age or older in available vaccination and hospitalization records. Table 13 shows the 5454 specimen results from 5437 routine newborn specimens along with the 17 confirmed positive SCID cases. For the confirmed positive SCID cases, the comparator was the reference tests results. In the initial round of testing the EnLite™ Neonatal TREC Kit identified all of the 17 confirmed SCID specimens as below the cut-off of