Alegria Flash SSA-60; Alegria Flash SSA-52; Alegria FLash SSB
Device Facts
| Record ID | K250814 |
|---|---|
| Device Name | Alegria Flash SSA-60; Alegria Flash SSA-52; Alegria FLash SSB |
| Applicant | Zeus Scientific |
| Product Code | OBE · Immunology |
| Decision Date | May 8, 2026 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5100 |
| Device Class | Class 2 |
Indications for Use
Alegria Flash SSA-52: The Alegria Flash SSA-52 kit uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to SSA-52 in human serum. It is intended for use as an aid in the diagnosis of Sjögren’s syndrome (SJS), Systemic Lupus Erythematosus (SLE), Systemic Sclerosis (SS), Polymyositis (PM), and Dermatomyositis (DM) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. Alegria Flash SSA-60: The Alegria Flash SSA-60 kit uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to SSA-60 in human serum. It is intended for use as an aid in the diagnosis of Sjögren’s syndrome (SS) and Systemic Lupus Erythematosus (SLE) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. Alegria Flash SSB: The Alegria Flash SSB kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to SSB (La) in human serum. It is intended for use as an aid in the diagnosis of Sjögren’s syndrome (SS) and Systemic Lupus Erythematosus (SLE) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument.
Device Story
Automated chemiluminescent immunoassay (CLIA) system for detection of IgG autoantibodies (SSA-52, SSA-60, SSB) in human serum; utilizes magnetic particles coated with recombinant or native antigens; performed on Alegria Flash instrument. Process: sample/diluent/beads incubation; wash; addition of acridinium ester-conjugated anti-human IgG; second incubation; wash; trigger solution addition; flash chemiluminescence measured by photomultiplier. Output: relative light units (RU); qualitative (SSA-52, SSA-60) or semi-quantitative (SSB) results. Used in clinical laboratories; operated by trained personnel. Results aid clinicians in diagnosing systemic autoimmune disorders alongside clinical findings.
Clinical Evidence
Clinical validation included 913 samples: Sjögren’s syndrome (N=134), SLE (N=178), SSc (N=124), PM (N=50), DM (N=50), and controls (N=376). SSA-52 sensitivity: 41.6% (SLE), 73.9% (SjS), 20.2% (SSc), 22.0% (PM), 26.0% (DM); specificity 95.7%. SSA-60 sensitivity: 51.7% (SLE), 77.6% (SjS); specificity 95.0%. SSB sensitivity: 20.2% (SLE), 51.5% (SjS); specificity 98.3%.
Technological Characteristics
Chemiluminescent immunoassay (CLIA) technology; utilizes the Alegria Flash instrument for automated processing; detects IgG antibodies to SSA-52, SSA-60, and SSB (La) in human serum.
Indications for Use
Indicated for qualitative (SSA-52, SSA-60) or semi-quantitative (SSB) detection of IgG antibodies in human serum to aid in the diagnosis of Sjögren’s syndrome, Systemic Lupus Erythematosus, Systemic Sclerosis, Polymyositis, and Dermatomyositis in conjunction with clinical findings. Prescription use only.
Regulatory Classification
Identification
An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues).
Submission Summary (Full Text)
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FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY ONLY
## I Background Information:
A 510(k) Number
K250814
B Applicant
ZEUS Scientific
C Proprietary and Established Names
- Alegria Flash SSA-60
- Alegria Flash SSA-52
- Alegria Flash SSB
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| OBE
LLL
PET | Class II | 21 CFR 866.5100 - Antinuclear Antibody
Immunological Test System | IM - Immunology |
## II Submission/Device Overview:
A Purpose for Submission:
New devices
B Measurand:
- Human anti-SSA-52 (anti-Ro52) IgG autoantibodies
- Human anti-SSA-60 (anti-Ro60) IgG autoantibodies
- Human anti-SSB IgG autoantibodies
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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K250814 - Page 2 of 20
C Type of Test:
Alegria Flash SSA-52: automated qualitative, chemiluminescent immunoassay (CLIA)
Alegria Flash SSA-60: automated qualitative, chemiluminescent immunoassay (CLIA)
Alegria Flash SSB: automated semi-quantitative, chemiluminescent immunoassay (CLIA)
III Intended Use/Indications for Use:
A Intended Use(s):
See Indications for Use below.
B Indication(s) for Use:
Alegria Flash SSA-52
The Alegria Flash SSA-52 kit uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to SSA-52 in human serum. It is intended for use as an aid in the diagnosis of Sjögren’s syndrome (SJS), Systemic Lupus Erythematosus (SLE), Systemic Sclerosis (SS), Polymyositis (PM), and Dermatomyositis (DM) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument.
Alegria Flash SSA-60
The Alegria Flash SSA-60 kit uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to SSA-60 in human serum. It is intended for use as an aid in the diagnosis of Sjögren’s syndrome (SS) and Systemic Lupus Erythematosus (SLE) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument.
Alegria Flash SSB
The Alegria Flash SSB kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to SSB (La) in human serum. It is intended for use as an aid in the diagnosis of Sjögren’s syndrome (SS) and Systemic Lupus Erythematosus (SLE) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument.
C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
D Special Instrument Requirements:
Zeus Alegria Flash instrument (K230863)
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IV Device/System Characteristics:
# A Device Description:
## Alegria Flash SSA-52
The Alegria Flash SSA-52 kit contains the following materials:
- One (1) Assay Cartridge containing bead suspension, sample diluent, and conjugate reagents.
- Bead suspension (5 mL): Magnetic particles coated with recombinant SSA-52 antigen in storage buffer containing Tween-20, bovine serum albumin, phosphate-buffered-saline, and <0.1% sodium azide. Ready to use.
- Sample diluent (10 mL): Phosphate-buffered-saline solution containing detergent, proteins, <0.1% sodium azide, and ProClin 300. Ready to use.
- Conjugate (10 mL): Anti-human IgG conjugated with Acridinium Ester in a buffered solution containing ProClin 300. Ready to use.
- One (1) Calibrator 1 (blue-capped vial) (0.5 mL): Human serum that contains anti-SSA-52 autoantibodies at levels above the cutoff value and <0.1% sodium azide. Ready to use. Assay and lot specific.
- One (1) Calibrator 2 (white-capped vial) (0.5 mL): Human serum containing anti-SSA-52 autoantibodies at levels near the cutoff value and <0.1% sodium azide. Ready to use. Assay and lot specific.
A separate Alegria Flash SSA-52 Control kit is sold separately. It contains:
- One (1) positive control ("CTRL +") (1.0 mL/vial): Human serum containing anti-SSA-52 antibodies at levels above the cutoff value and <0.1% sodium azide. Ready to use and assay specific.
- One (1) negative control ("CTRL -") (1.0 mL/vial): Human serum containing anti-SSA-52 antibodies at levels below the cutoff value and <0.1% sodium azide. Ready to use and assay specific.
## Alegria Flash SSA-60
The Alegria Flash SSA-60 kit contains the following materials:
- One (1) Assay Cartridge containing bead suspension, sample diluent, and conjugate reagents.
- Bead suspension (0.5 mL): Magnetic particles coated with recombinant SSA-60 antigen in storage buffer containing Tween-20, bovine serum albumin, phosphate-buffered-saline, and <0.1% sodium azide. Ready to use.
- Sample diluent (10 mL): Phosphate-buffered-saline solution containing detergent, proteins, <0.1% sodium azide, and ProClin 300. Ready to use.
- Conjugate (10 mL): Anti-human IgG conjugated with Acridinium Ester in a buffered solution containing ProClin 300. Ready to use.
- One (1) Calibrator 1 (blue-capped vial) (0.5 mL): Human serum that contains anti-SSA-60 autoantibodies at levels above the cutoff value and <0.1% sodium azide. Ready to use. Assay and lot specific.
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- One (1) Calibrator 2 (white-capped vial) (0.5 mL): Human serum containing anti-SSA-60 autoantibodies at levels near the cutoff value and <0.1% sodium azide. Ready to use. Assay and lot specific.
A separate Alegria Flash SSA-52 Control kit is sold separately. It contains:
- One (1) positive control ("CTRL +") (0.1 mL/vial): Human serum containing anti-SSA-60 antibodies at levels above the cutoff value and <0.1% sodium azide. Ready to use and assay specific.
- One (1) negative control ("CTRL -") (1.0 mL/vial): Human serum containing anti-SSA-60 antibodies at levels below the cutoff value and <0.1% sodium azide. Ready to use and assay specific.
## Alegria Flash SSB
The Alegria Flash SSB kit contains the following materials:
- One (1) Assay Cartridge containing bead suspension, sample diluent, and conjugate reagents.
- Bead suspension (5 mL): Magnetic particles that are coated with SSB (La) antigen in storage buffer containing Tween-20, bovine serum albumin, phosphate-buffered-saline, and <0.1% sodium azide. Ready to use.
- Sample diluent (10 mL): Phosphate-buffered-saline solution containing detergent, proteins, <0.1% sodium azide, and ProClin 300. Ready to use.
- Conjugate (10 mL): Anti-human IgG that is conjugated with Acridinium Ester in a buffered solution containing ProClin 300. Ready to use.
- One (1) Calibrator 1 (blue-capped vial) (0.5 mL): Human serum containing anti-SSB autoantibodies at levels above the cutoff value and <0.1% sodium azide. Ready to use. Assay and lot specific.
- One (1) Calibrator 2 (white-capped vial) (0.5 mL): Human serum containing anti-SSB autoantibodies at levels near the cutoff value and <0.1% sodium azide. Ready to use. Assay and lot specific.
A separate Alegria Flash SSB Control kit is sold separately. It contains:
- One (1) positive control ("CTRL +") (1.0 mL/vial): Human serum containing anti-SSB antibodies at levels above the cutoff value and <0.1% sodium azide. Ready to use and assay specific.
- One (1) negative control ("CTRL -") (1.0 mL/vial): Human serum containing anti-SSB antibodies at levels below the cutoff value and <0.1% sodium azide. Ready to use and assay specific.
## B Principle of Operation:
The Alegria Flash SSA-52, SSA-60, SSB assay kits are designed to detect human anti-SSA-52, SSA-60, and SSB IgG autoantibodies, in human serum. The Alegria Flash SSA-52, SSA-60, SSB test procedures involve three main steps. Sample diluent, test sera, and antigen-coated magnetic particles are added to a reaction cuvette. During the initial incubation, autoantibodies specific to SSA-52, SSA-60, or SSB present in the serum will bind to the immobilized antigens. The beads are then washed to remove unbound antibodies and other serum components. An
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acridinium ester-conjugated anti-human IgG solution is then added to the reaction cuvette. During the second incubation, the conjugate reacts with IgG autoantibodies that are immobilized on the magnetic particles in step 1. The beads are then washed to remove unbound conjugate. Two trigger solutions are added to the cuvette containing immobilized acridinium ester conjugate, causing a flash chemiluminescence reaction to occur. The light signal released by the chemiluminescence reaction is measured by a photomultiplier within the Alegria Flash analyzer and the signal is reported as relative light units (RU). RU values above or equal to the cut-off value of 100 RU are indicative of the presence of anti-SSA-52, anti-SSA-60, or anti-SSB antibodies within the original serum sample.
V Substantial Equivalence Information:
A Predicate Device Name(s):
QUANTA Flash Ro52
QUANTA Flash Ro60
AtheNA Multi-Lyte ANA-II Plus Test System (SS-B)
B Predicate 510(k) Number(s):
K141655
K141328
K021103
C Comparison with Predicate(s):
Alegria Flash SS-A 52
| Device & Predicate Device(s): | K250814 (Candidate Device) | K141655 (Predicate Device) |
| --- | --- | --- |
| Device Trade Name | Alegria Flash SSA-52 | QUANTA Flash SSA-52 |
| General Device Characteristic Similarities | | |
| Detection Principle | Chemiluminescent immunoassay | Same |
| Sample Matrix | Serum | Same |
| Antigen | Recombinant Ro52 | Same |
| General Device Characteristic Differences | | |
| Intended Use/Indications For Use | The Alegria Flash SSA-52 kit uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to SSA-52 in human serum. It is intended for use as an aid in the diagnosis of Sjögren's syndrome (SJS), Systemic Lupus Erythematosus (SLE), Systemic | QUANTA Flash Ro52 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro52 autoantibodies in human serum. The presence of anti-Ro52 autoantibodies, in conjunction with clinical findings and other |
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| | Sclerosis (SS), Polymyositis (PM), and Dermatomyositis (DM) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. | laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus, Sjögren’s Syndrome, Systemic Sclerosis, Idiopathic Inflammatory Myopathies. |
| --- | --- | --- |
| Type of Test | Qualitative | Semi-quantitative |
| Instrumentation | Alegria Flash | QUANTA Flash |
| Assay Units | Relative light units (RU) | Chemiluminescent units (CU) |
| Analytical Measuring Interval | Not applicable | 2.3 CU – 1685.3 CU |
| Cut-off | 100 RU | 20 CU |
Alegria Flash SS-A 60
| Device & Predicate Device(s): | K250814
(Candidate Device) | K141328
(Predicate Device) |
| --- | --- | --- |
| Device Trade Name | Alegria Flash SSA-60 | QUANTA Flash SSA-60 |
| General Device Characteristic Similarities | | |
| Detection Principle | Chemiluminescent immunoassay | Same |
| Sample Matrix | Serum | Same |
| General Device Characteristic Differences | | |
| Intended Use/Indications For Use | The Alegria Flash SSA-60 kit uses chemiluminescent immunoassay (CLIA) technology for the qualitative detection of IgG antibodies to SSA-60 in human serum. It is intended for use as an aid in the diagnosis of Sjögren’s syndrome (SS) and Systemic Lupus Erythematosus (SLE) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. | QUANTA Flash Ro60 is a chemiluminescent immunoassay for the semi-quantitative determination of IgG anti-Ro60 autoantibodies in human serum. The presence of anti-Ro60 autoantibodies, in conjunction with clinical findings and other laboratory tests, is an aid in the diagnosis of Systemic Lupus Erythematosus and Sjögren’s Syndrome. |
| Instrumentation | Alegria Flash | QUANTA Flash |
| Assay Units | Relative light units (RU) | Chemiluminescent units (CU) |
| Antigen | Purified Native SSA-60 | Recombinant SSA-60 |
| Type of Test | Qualitative | Semi-quantitative |
| Analytical Measuring Interval | Not applicable | 4.9 CU – 1374.8 CU |
| Cut-off | 100 RU | 20 CU |
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Alegria Flash SSB
| Device & Predicate Device(s): | K250814 (Candidate Device) | K021103 (Predicate Device) |
| --- | --- | --- |
| Device Trade Name | Alegria Flash SSB | Zeus AtheNA Multi-Lyte SSB |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | The Alegria Flash SSB kit uses chemiluminescent immunoassay (CLIA) technology for the semi-quantitative measurement of IgG antibodies to SSB (La) in human serum. It is intended for use as an aid in the diagnosis of Sjögren's syndrome (SS) and Systemic Lupus Erythematosus (SLE) in conjunction with other laboratory and clinical findings. The test must be performed on the Alegria Flash instrument. | The ZEUS AtheNA Multi-Lyte ANA-II Plus Test System is intended for the semi-quantitative detection of IgG class antibody to eight separate analytes (SSA, SSB, Sm,RNP, Scl-70, Jo-1, Centromere B, and Histone) in human serum, the quantitative detection of IgG class antibody to dsDNA in human serum, and the qualitative detection of IgG class antibody to ANA. The test system is intended to be used as an aid in the diagnosis of various systemic autoimmune disorders. This test is for In Vitro Diagnostic use. |
| Type of Test | Semi-quantitative | Same |
| Sample Matrix | Serum | Same |
| General Device Characteristic Differences | | |
| Detection Principle | Chemiluminescent immunoassay | Luminex-based, multiplex Immunoassay |
| Instrumentation | Alegria Flash | AtheNA Multi-Lyte platform |
| Antigen | Recombinant SSB | Purified SSB |
| Analytical Measuring Interval | 20.2 – 526.2 RU | Not specified |
| Cut-off | 100 RU | < 100 Units Negative 100 – 120 Units Equivocal > 120 Units Positive |
# VI Standards/Guidance Documents Referenced:
The following Clinical and Laboratory Standards Institute (CLSI) guidelines were used:
- CLSI EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition
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- CLSI EP06-Ed2, Evaluation of the Linearity of Quantitative Measurement Procedures – Second Edition
- CLSI EP07-Ed3 – Interference Testing in Clinical Chemistry
- CLSI EP09c 3rd Edition, Measurement Procedure Comparison and Bias Estimation Using Patient Samples
- CLSI EP12-Ed3 – Evaluation of Qualitative, Binary Output Examination Performance
- CLSI EP17-A2 – Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures
- CLSI EP25-Ed2 – Evaluation of Stability of In Vitro Medical Laboratory Test Reagents
- CLSI EP28-A3c – Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory
## VII Performance Characteristics (if/when applicable):
### A Analytical Performance:
#### 1. Precision/Reproducibility:
Precision and reproducibility of the Alegria Flash SSA-52, Alegria Flash SSA-60, and Alegria Flash SSB were evaluated in accordance with the CLSI guideline EP05-A3. The samples panels were prepared by mixing positive native serum samples with negative serum samples.
#### Within-Lab Precision:
To evaluate within-laboratory precision of the assays, a variable number of samples containing various concentrations of antibodies were assayed in duplicate, twice a day, for 20 days, for a total of 80 measurements per sample, using one reagent lot at one laboratory site, by one operator.
- For the qualitative Alegria Flash SSA-52 and Alegria Flash SSA-60 assays, the percent of positive (% Pos), with 95% confidence interval (CI), for each sample was calculated.
- For the semi-quantitative Alegria Flash SSB assay, the data was analyzed using Analyze-it for Excel software by analysis of variance (ANOVA) methods and the within-run (repeatability), between-run, between-day, and within-laboratory precision were determined.
The results are summarized in the following tables:
Alegria Flash SSA-52:
| Sample | Expected Result | Relative Units (RU) | | Positive | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Mean | Range | n(+)/N | % Pos. | (95% CI) |
| 1 | Positive | 403.1 | 361.8–441.6 | 80/80 | 100 | (95.4–100) |
| 2 | Positive | 333.7 | 286.0–462.1 | 80/80 | 100 | (95.4–100) |
| 3 | Positive | 250.6 | 198.4–370.3 | 80/80 | 100 | (95.4–100) |
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| Sample | Expected Result | Relative Units (RU) | | Positive | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Mean | Range | n(+)/N | % Pos. | (95% CI) |
| 4 | Positive | 162.8 | 123.2–209.5 | 80/80 | 100 | (95.4–100) |
| 5 | Positive | 101.2 | 74.4–132.5 | 80/80 | 100 | (95.4–100) |
| 6 | Negative | 85.2 | 73.4–99.4 | 47/80 | 58.8 | (47.8–68.9) |
| 7 | Negative | 66.8 | 49.0–93.4 | 0/80 | 0.0 | (0.0–4.6) |
| 8 | Negative | 35.5 | 28.4–42.9 | 0/80 | 0.0 | (0.0–4.6) |
Alegria Flash SSA-60:
| Sample | Expected Result | Relative Units (RU) | | Positive | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Mean | Range | n(+)/N | % Pos. | (95% CI) |
| 1 | Positive | 365.6 | 271.8–425.7 | 80/80 | 100 | (95.4–100) |
| 2 | Positive | 269.2 | 213.4–317.5 | 80/80 | 100 | (95.4–100) |
| 3 | Positive | 168.4 | 125.6–202.3 | 80/80 | 100 | (95.4–100) |
| 4 | Negative | 98.0 | 77.6–111.2 | 36/80 | 45.0 | (34.6–55.9) |
| 5 | Negative | 88.4 | 64.2–107.3 | 6/80 | 7.5 | (3.5–15.4) |
| 6 | Negative | 65.8 | 47.1–79.2 | 0/80 | 0.0 | (0.0–4.6) |
| 7 | Negative | 27.8 | 20.0–35.5 | 0/80 | 0.0 | (0.0–4.6) |
Alegria Flash SSB
| | | Within-Run | | Between-Run | | Between-Day | | Within-Laboratory | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (RU) | SD (RU) | %CV | SD (RU) | %CV | SD (RU) | %CV | SD (RU) | %CV |
| 1 | 399.3 | 16.3 | 4.1 | 0.0 | 0.0 | 21.8 | 5.5 | 27.2 | 6.8 |
| 2 | 238.6 | 7.5 | 3.2 | 4.6 | 1.9 | 10.4 | 4.4 | 13.7 | 5.7 |
| 3 | 118.7 | 6.1 | 5.1 | 0.9 | 0.7 | 5.2 | 4.4 | 8.1 | 6.8 |
| 4 | 92.3 | 3.2 | 3.5 | 2.8 | 3.0 | 6.4 | 7.0 | 7.7 | 8.3 |
| 5 | 71.1 | 2.8 | 3.9 | 0.7 | 1.0 | 3.3 | 4.7 | 4.4 | 6.2 |
| 6 | 41.9 | 2.3 | 5.4 | 0.5 | 1.3 | 1.5 | 3.6 | 2.8 | 6.6 |
# Site-to-Site Reproducibility:
The reproducibility of the Alegria Flash assays was conducted at three sites using a variable number of samples containing various concentrations of antibodies. Samples were assayed in quintuplicate, once a day, for five days to generate 25 data points per sample per site using one reagent lot and a total of 75 replicates per sample. Instrument variables were nested within the multiple site component – i.e., a different instrument was used at each of the three sites.
- For the qualitative Alegria Flash SSA-52 and Alegria Flash SSA-60 assays, the percent of positive (% Pos), with $95\%$ confidence interval (CI) for all sites combined, for each sample was calculated.
- For the semi-quantitative Alegria Flash SSB assay, the data was analyzed using Analyze-it for Excel software by analysis of variance (ANOVA) methods and the within-run
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(repeatability), between-run, between-day, and within-laboratory precision were determined.
The results are summarized in the following tables:
## Alegria Flash SSA-52
| Sample | Expected Result | Total (across sites) | | | | Site 1 | | Site 2 | | Site 3 | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Mean (RU) | Range (RU) | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos |
| 1 | Positive | 403.9 | 361.2–436.4 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 2 | Positive | 239.9 | 208.2–273.9 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 3 | Positive | 129.2 | 116.6–150.0 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 15/25 | 60 |
| 4 | Positive | 102.9 | 91.2–121.0 | 47/75 | 62.7 | 8/25 | 32 | 24/25 | 86 | 25/25 | 100 |
| 5 | Negative | 72.7 | 61.2–84.8 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 |
| 6 | Negative | 34.1 | 27.0–44.0 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 |
## Alegria Flash SSA-60
| Sample | Expected Result | Total (across sites) | | | | Site 1 | | Site 2 | | Site 3 | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Mean (RU) | Range (RU) | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos |
| 1 | Positive | 399.3 | 243.1–464.8 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 2 | Positive | 241.9 | 190.2–275.6 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 3 | Positive | 130.5 | 103.6–149.9 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 15/25 | 60 |
| 4 | Negative | 93.6 | 60.0–109.7 | 16/75 | 21.3 | 7/25 | 28 | 2/25 | 8 | 7/25 | 28 |
| 5 | Negative | 75.8 | 55.5–86.9 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 |
| 6 | Negative | 41.5 | 30.2–50.1 | 0/75 | 0 | 0/25 | 0 | 0/25 | 0 | 0/25 | 0 |
## Alegria Flash SSB
| | | Within-Run | | Between-Day | | Between-Site/Instrument | | Reproducibility | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (RU) | SD (RU) | %CV | SD (RU) | %CV | SD (RU) | %CV | SD (RU) | %CV |
| 1 | 406.7 | 17.7 | 4.3 | 21.7 | 5.3 | 2.0 | 0.5 | 28.0 | 6.9 |
| 2 | 245.3 | 11.3 | 4.6 | 8.7 | 3.6 | 8.2 | 3.3 | 16.4 | 6.7 |
| 3 | 122.7 | 5.5 | 4.5 | 3.9 | 3.2 | 5.1 | 4.2 | 8.5 | 6.9 |
| 4 | 93.3 | 3.3 | 3.6 | 3.7 | 4.0 | 5.2 | 5.6 | 7.2 | 7.7 |
| 5 | 74.7 | 3.6 | 4.9 | 2.4 | 3.2 | 6.5 | 8.8 | 7.9 | 10.5 |
| 6 | 48.1 | 2.8 | 5.8 | 1.7 | 3.5 | 4.6 | 9.6 | 5.7 | 11.7 |
## Lot-to-Lot Imprecision
To evaluate the between-lot imprecision of the Alegria Flash, a variable number of samples containing various concentrations of antibodies were assayed in quintuplicate, once a day, for five days, using three reagent lots, using one instrument, for a total of 75 replicates per sample.
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- For the qualitative Alegria Flash SSA-52 and Alegria Flash SSA-60 assays, the percent of positive (% Pos), with 95% confidence interval (CI) for all lots combined, for each sample was calculated.
- For the semi-quantitative Alegria Flash SSB assay, the data was analyzed using Analyze-it for Excel software by analysis of variance (ANOVA) methods and the within-run (repeatability), between-run, between-day, and within-laboratory precision were determined.
Alegria Flash SSA-52:
| Sample | Expected Result | Total (across lots) | | | | Lot 1 | | Lot 2 | | Lot 3 | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Mean (RU) | Range (RU) | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos |
| 1 | Positive | 414.6 | 361.2–483.1 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 2 | Positive | 243.8 | 208.2–279.1 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 3 | Positive | 135.6 | 121.0–150.9 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 4 | Positive | 110.0 | 97.7–121.0 | 73/75 | 97.3 | 24/25 | 96 | 25/25 | 100 | 24/25 | 96.0 |
| 5 | Negative | 79.9 | 67.8–87.4 | 0/75 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 |
| 6 | Negative | 38.1 | 30.7–44.4 | 0/75 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 |
Alegria Flash SSA-60:
| Sample | Expected Result | Total (across lots) | | | | Lot 1 | | Lot 2 | | Lot 3 | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Mean (RU) | Range (RU) | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos | n(+)/N | % Pos |
| 1 | Positive | 409.3 | 308.1–463.3 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 2 | Positive | 250.3 | 218.3–281.7 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 3 | Positive | 141.4 | 110.4–158.9 | 75/75 | 100 | 25/25 | 100 | 25/25 | 100 | 25/25 | 100 |
| 4 | Positive | 105.7 | 86.5–122.7 | 56/75 | 74.7 | 7/25 | 28 | 24/25 | 96 | 25/25 | 100 |
| 5 | Negative | 85.7 | 74.4–99.3 | 0/75 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 |
| 6 | Negative | 42.9 | 32.2–51.1 | 0/75 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 | 0/25 | 0.0 |
Alegria Flash SSB:
| | | Within-Run | | Between-Day | | Between-Lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Mean (RU) | SD (RU) | %CV | SD (RU) | %CV | SD (RU) | %CV | SD (RU) | %CV |
| 1 | 389.9 | 19.4 | 5.0 | 18.2 | 4.7 | 0 | 0 | 26.6 | 6.8 |
| 2 | 230.6 | 10.0 | 4.3 | 9.0 | 3.9 | 1.4 | 0.6 | 13.5 | 5.9 |
| 3 | 117.1 | 5.6 | 4.8 | 4.9 | 4.1 | 2.2 | 1.9 | 7.8 | 6.6 |
| 4 | 87.7 | 2.8 | 3.2 | 4.0 | 4.6 | 2.5 | 2.9 | 5.5 | 6.3 |
| 5 | 68.9 | 2.4 | 3.5 | 3.1 | 4.5 | 2.7 | 4.0 | 4.8 | 6.9 |
| 6 | 43.3 | 2.5 | 5.9 | 2.2 | 5.2 | 2.3 | 5.2 | 4.1 | 9.4 |
2. Linearity:
The linearity of the semi-quantitative Alegria Flash SSB analytical measuring range was evaluated by following CLSI EP06-Ed2. Various samples were prepared as overlapping dilution series by mixing human serum samples with high antibody concentrations and
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samples with low antibody concentrations to cover portions of the analytical measuring interval (AMI) of each analyte. Each sample was tested in five replicates and the results from each sample were analyzed separately using a weighted least squares regression analysis. All dilutions included in the sample regressions were within $\pm 10\%$ deviation from the linearity. The results are summarized below.
# Alegria Flash SSB:
| Sample | Test Range (RU) | Slope (95% CI) | R2 | Range of Linearity Deviations |
| --- | --- | --- | --- | --- |
| 1 | 172.8 – 526.2 | 0.96 (0.90 – 1.02) | 1.00 | -3.7% – 3.2% |
| 2 | 97.5 – 245.3 | 0.99 (0.85 – 1.14) | 0.98 | -4.4% – 6.2% |
| 3 | 20.2 – 99.9 | 1.01 (0.96 – 1.05) | 1.00 | -5.4% – 4.1 |
The data supports a linearity of the claimed analytical measuring interval (AMI) of 20.2-526.2 RU for the Alegria Flash SSA-60 assay.
The Alegria Flash SSB assay does not have an extended measuring range for samples above the AMI. Samples exceeding the AMI are reported as "above assay" in the result report. The package insert instructs users to report the result as $>526.2$ RU.
# 3. Analytical Specificity/Interference:
An interference study was performed based on the recommendations contained in CLSI EP07-A2. Two human serum specimens - one positive specimen at $\sim 125$ RU and one negative specimen at $\sim 75$ RU, were assessed as vehicle-control or interferent-spiked contrived specimens in five replicates using the Alegria Flash SSA-52, Alegria Flash SSA-60, and Alegria Flash SSB assays. The percent recovery for each sample that was spiked with the potential interfering substance was calculated by comparing its result to that of the corresponding control sample without the interfering substance. The following endogenous and exogenous substances showed $< \pm 15\%$ recovery than the control:
| Endogenous Substances | | Exogenous Substances | |
| --- | --- | --- | --- |
| Interferent | Testing Concentration | Interferent | Testing Concentration |
| Bilirubin | 0.15 mg/mL | Ibuprofen | 0.219 mg/ml |
| Cholesterol | 2.2 mg/mL | Hydroxychloroquine | 0.024 mg/mL |
| Triglycerides | 2.5 mg/mL | Prednisone | 0.000099 mg/mL |
| Albumin | 52 mg/mL | Azathioprine | 0.00258 mg/mL |
| Hemoglobin | 200 mg/dL | Diltiazem | 0.0009 mg/mL |
| RF | 400 U/mL | Rituximab | 2 mg/mL |
| | Methotrexate | 1.36 mg/mL | |
| | | Enalapril | 0.000819 mg/mL |
| | | Omeprazole | 0.0084 mg/mL |
| | | Losartan | 0.000306 mg/ml |
| | | Atenolol | 0.009 mg/mL |
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| Endogenous Substances | | Exogenous Substances | |
| --- | --- | --- | --- |
| Interferent | Testing Concentration | Interferent | Testing Concentration |
| | Erythromycin | 0.138 mg/mL | |
| | | Amoxicillin | 0.054 mg/mL |
| | | Ranitidine | 0.0105 mg/mL |
| | | Furosemide | 0.0159 mg/mL |
| | | Alendronate | 0.000034 mg/mL |
| | | Atorvastatin | 0.00075 mg/mL |
Reference Sera:
Selected Antinuclear Antibodies (ANA) IUIS Reference Standards samples, previously known as the CDC (Center for Disease Controls and Prevention) ANA Reference Panel, were tested in singlicate using one reagent lot of the three Alegria Flash assays to illustrate the analytical specificity of the assay. The results are outlined below.
| Panel ID | CDC Description | Alegria Flash Assay Result | | |
| --- | --- | --- | --- | --- |
| | | SSA-52 | SSA-60 | SSB |
| ANA 01 | ANA Homogenous Positive/ anti-native DNA | Negative | Negative | Negative |
| ANA 02 | ANA Speckled Positive/anti-SSB/La | Positive | Positive | Positive |
| ANA 03 | ANA Speckled Positive/ anti-U1 RNP, SSB/La, SSA/Ro | Negative | Positive | Positive |
| ANA 04 | anti-U1 RNP Positive | Negative | Negative | Negative |
| ANA 06 | ANA Nucleolar (U3RNP) Positive | Negative | Negative | Negative |
| ANA 07 | anti-SSA Ro Positive | Positive | Positive | Negative |
| ANA 08 | ANA Centromere Positive | Negative | Negative | Negative |
| ANA 09 | ANA anti-Scl70 Positive | Negative | Negative | Negative |
| ANA 10 | anti Jo-1 Positive | Positive | Negative | Negative |
| ANA 11 | anti-PM/Scl Positive | Negative | Negative | Negative |
| ANA 12 | anti-Ribosomal P Positive | Negative | Negative | Negative |
| ANA 15 | anti-MPO Positive | Negative | Negative | Negative |
| ANA 16 | anti-PR3 Positive | Negative | Negative | Negative |
4. Assay Reportable Range:
Not applicable for the qualitative Alegria Flash SSA-52 and Alegria Flash SSA-60.
The assay reportable range is the same as the analytical measuring intervals (AMI) for the Alegria Flash SSB assay: 20.2 - 526.2 RU
5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
Traceability
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There are no international reference standards for anti-SSA-52, anti-SSA-60, and anti-SSB antibodies that allow metrological standardization of the assays. Calibrator and control values are directly traceable to in-house reference materials that are used to create the master curves for each Alegria Flash assay lot.
**Stability:**
**Reagent stability:**
**Shelf-life:** The reagent stability studies were designed and conducted following CLSI EP25-Ed2. To assess the shelf-life stability of the Alegria Flash ENA Screen kits, serum sample panels were assessed in triplicate on three lots of Alegria Flash SSA-52, Alegria Flash SSA-60, and Alegria Flash SSB kits at various time points. The results support that the assay kits are stable when stored unopened at 2-8°C for up to six months.
**On-board stability:** The onboard storage stability of the three Alegria Flash kits was determined by testing three serum samples and two assay-specific Alegria Flash controls using the Alegria Flash reagents stored opened within the Alegria Flash’s refrigerated reagent bay. The results of the study support that Alegria Flash SSA-52, Alegria Flash SSA-60, and Alegria Flash SSB ENA reagents are stable when stored onboard or at 2-8°C for four weeks.
**Sample stability:**
The sample stability studies were designed and conducted following CLSI EP25-Ed2. To assess the stability of patient samples, two serum samples – a low positive and a high negative – were stored up to 15 days at 2-8°C or at room temperature. The samples were then assessed using three replicates and the percent recovery in comparison to the unmanipulated, baseline sample was determined for each storage condition. The results support that patient serum samples are stable when stored for up to 14 days at 2-8°C or at room temperature.
To determine the stability of patient samples subjected to multiple freeze/thaw cycles, two serum samples – a low positive and a high negative – were subjected to six rounds of freeze/thaw cycles, consisting of ≥12 hours of storage at -20°C and a subsequent thaw. The samples were assessed for each condition using three replicates and the percent recovery in comparison to the unmanipulated, baseline sample was determined for each freeze/thaw cycle. The results support that patient serum samples are stable when frozen at -20°C and thawed for a maximum of five times.
6. **Detection Limit:**
Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) for the Alegria Flash SSB assay were determined by following CLSI EP17-A2.
**LoB:** To determine the LoB, four analyte-free blank samples were run in 20 replicates, once a day for three days using two reagent lots, for a total of 60 data points per sample and lot (480 total replicates). The LoB was determined for each analyte, on each reagent lot separately, at the 95th percentile using the non-parametric method for all analyses. The higher LoB result between the two lots was selected for the final LoB value. The LoB of the assay is 17.4 RU.
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LoD: To determine the LoD for each analyte, four low level samples were assayed in 20 replicates, for three days using two reagent lots, to generate a total of 60 data points for each sample on each reagent lot. The LoD was determined separately for each sample on each reagent lot and the highest LoD result was determined for the final LoD value. The LoD of the assay is 20.1 RU.
LoQ: To determine the LoQ for each analyte, the four LoD samples and an additional four low-level samples greater than the LoD were run in 20 replicates, for three days using two reagent lots, to generate 60 data points for each sample on each reagent lot. The LoQ was determined separately for each sample on each reagent lot by calculating the total imprecision of each sample. The LoQ was defined as the lowest concentration level that meets the within-laboratory imprecision of ≤15% for each lot. The LoQ of the assay is 21.1 RU.
7. Assay Cut-Off:
The assays' cut-offs are 100 RU.
B Comparison Studies:
1. Method Comparison with Predicate Device:
Clinically characterized samples were tested on the Alegria Flash assays and their predicate devices. Positive percent agreement (PPA) and negative percent agreement (NPA), with 95% confidence intervals were calculated for each analyte comparison. For the qualitative Alegria Flash SSA-52 and Alegria Flash SSA-60 assays, all values were included in the PPA and NPA determination. For the semi-quantitative SS-B assay, values that were outside of the measuring ranges of either assay were excluded from the calculations. The following tables summarize the assays' method comparison study results:
Alegria Flash SSA-52:
| | Predicate | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| Alegria Flash SSA-52 | Positive | 57 | 6 | 163 |
| | Negative | 0 | 54 | 54 |
| | Total | 57 | 60 | 117 |
PPA 100% (95%CI 93.7 - 100.0)
NPA 90% (95%CI 79.9 - 95.3)
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Alegria Flash SSA-60:
| | Predicate | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| Alegria Flash SSA-52 | Positive | 57 | 6 | 163 |
| | Negative | 0 | 54 | 54 |
| | Total | 57 | 60 | 117 |
PPA 100% (95%CI 93.7 – 100.0)
NPA 90% (95%CI 79.9 – 95.3)
Alegria Flash SSB:
| | Predicate SSB | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Positive | Equivocal | Negative | Total |
| Alegria Flash SSB | Positive | 59 | 1 | 7 | 67 |
| | Negative | 6 | 4 | 313 | 323 |
| | Total | 65 | 5 | 320 | 390 |
Equiv as Neg
PPA 90.8% (95%CI 81.3 – 95.7)
NPA 97.5% (95%CI 95.2 – 98.8)
Equiv as Pos
PPA 85.7% (95%CI 75.7 – 92.0)
NPA 97.8% (95%CI 95.6 – 98.9)
2. Matrix Comparison:
Not applicable.
C Clinical Studies:
1. Clinical Sensitivity and Specificity:
A cohort of characterized samples were used to validate the clinical performance of the Alegria Flash SSA-52, Alegria Flash SSA-60, and Alegria Flash SSB assays. The clinical validation study included 913 total samples including samples from patients with Sjögren’s syndrome (SjS, N=134), systemic lupus erythematosus (SLE, N=178), systemic sclerosis (SSc, N=124)¹, polymyositis (PM, N=50), dermatomyositis (DM, N=50) and control samples (N=376) from patients with various types of autoimmune and infectious diseases. The performance summarized below is organized by assay:
¹ The SSc cohort consisted of 70 samples diagnosed with SSc using the 2013 EULAR/ACR Classification Criteria but were not further categorized and 54 samples diagnosed with limited cutaneous systemic sclerosis (lcSSc, formerly known as CREST syndrome).
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Alegria Flash SSA-52:
| | Diagnosis | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SLE | SjS | SSc | PM | DM | Controls | Total |
| Alegria Flash SSA-52 | Positive | 74 | 99 | 25 | 11 | 13 | 16 | 238 |
| | Negative | 104 | 35 | 89 | 39 | 37 | 360 | 674 |
| | Total | 178 | 134 | 124 | 50 | 50 | 376 | 912 |
SLE Sensitivity $41.6\%$ (95%CI 34.6 - 48.9%)
SjS Sensitivity 73.9% (95%CI 65.8 - 80.6%)
SSc Sensitivity 20.2% (95%CI 14.0-28.1%)
PM Sensitivity 22.0% (95%CI 12.8 - 35.2%)
DM Sensitivity 26.0% (95%CI 15.9 - 39.6%)
Specificity 95.7% (95%CI 93.2 - 97.4%)
| SSA-52 Distribution of clinical study samples and antibody positivity rates: | | | |
| --- | --- | --- | --- |
| Diagnostic Group | N | SSA-52 Positive | |
| | | n | (%) |
| Target conditions | | | |
| Systemic lupus erythematosus (SLE) | 178 | 74 | 41.6 |
| Sjögren's Syndrome (SjS) | 134 | 99 | 73.9 |
| Systemic sclerosis (SSc) | 124 | 25 | 20.2 |
| Polymyositis (PM) | 50 | 11 | 22.0 |
| Dermatomyositis (DM) | 50 | 13 | 26.0 |
| Differential diagnosis controls | | | |
| Mixed Connective Tissue Disease | 20 | 4 | 20.0 |
| Autoimmune Hepatitis | 30 | 2 | 6.7 |
| Antiphospholipid Syndrome | 20 | 0 | 0 |
| Cancer | 19 | 0 | 0 |
| Celiac Disease | 24 | 0 | 0 |
| Drug Induced Lupus | 5 | 2 | 40.0 |
| Fibromyalgia | 20 | 0 | 0 |
| Crohn's Disease | 30 | 0 | 0 |
| Ulcerative Colitis | 29 | 0 | 0 |
| HBV | 17 | 2 | 11.8 |
| HIV | 12 | 0 | 0 |
| HCV | 10 | 1 | 10.0 |
| HSV | 10 | 0 | 0 |
| Primary Biliary Cholangitis | 30 | 1 | 3.3 |
| Rheumatoid Arthritis | 20 | 2 | 10.0 |
| Vasculitis (Undifferentiated) | 6 | 0 | 0 |
| Vasculitis (ANCA Associated) | 5 | 0 | 0 |
| Vasculitis (Large/Med Vessel) | 10 | 0 | 0 |
| Atrophic Gastritis | 20 | 0 | 0 |
| Graves' Disease | 20 | 0 | 0 |
| Hashimoto's Thyroiditis | 19 | 2 | 10.5 |
| Total Controls | 376 | 16 | 4.3% |
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Alegria Flash SSA-60:
| | Diagnosis | | | | |
| --- | --- | --- | --- | --- | --- |
| | | SLE | SjS | Controls | Totals |
| Alegria Flash SSA-60 | Positive | 92 | 104 | 30 | 226 |
| | Negative | 86 | 30 | 570 | 686 |
| | Total | 178 | 134 | 600 | 912 |
| SLE | Sensitivity | 51.7% | | (95%CI 44.4 – 58.9) | |
| SjS | Sensitivity | 77.6% | | (95%CI 69.8 – 83.8) | |
| | Specificity | 95.0% | | (95%CI 93.0 – 96.5) | |
| SSA-60Distribution of clinical study samples and antibody positivity rates: | | | | | |
| Diagnostic Group | N | SSA-60 Positive | | | |
| | | n | (%) | | |
| Target conditions | | | | | |
| Systemic lupus erythematosus (SLE) | | 178 | 92 | 51.7 | |
| Sjögren’s Syndrome (SjS) | | 134 | 104 | 77.6 | |
| Differential diagnosis controls | | | | | |
| Systemic sclerosis (SSc) | | 124 | 9 | 7.3 | |
| Polymyositis (PM) | | 50 | 3 | 6.0 | |
| Dermatomyositis (DM) | | 50 | 4 | 8.0 | |
| Mixed Connective Tissue Disease | | 20 | 2 | 10.0 | |
| Autoimmune Hepatitis | | 30 | 2 | 6.7 | |
| Antiphospholipid Syndrome | | 20 | 0 | 0 | |
| Cancer | | 19 | 0 | 0 | |
| Celiac Disease | | 24 | 0 | 0 | |
| Drug Induced Lupus | | 5 | 2 | 40.0 | |
| Fibromyalgia | | 20 | 1 | 5.0 | |
| Crohn's Disease | | 30 | 0 | 0 | |
| Ulcerative Colitis | | 29 | 0 | 0 | |
| HBV | | 17 | 1 | 5.9 | |
| HIV | | 12 | 0 | 0 | |
| HCV | | 10 | 0 | 0 | |
| HSV | | 10 | 0 | 0 | |
| Primary Biliary Cholangitis | | 30 | 1 | 3.3 | |
| Rheumatoid Arthritis | | 20 | 1 | 5.0 | |
| Vasculitis (Undifferentiated) | | 6 | 1 | 16.7 | |
| Vasculitis (ANCA Associated) | | 5 | 0 | 0 | |
| Vasculitis (Large/Med Vessel) | | 10 | 0 | 0 | |
| Atrophic Gastritis | | 20 | 0 | 0 | |
| Graves’ Disease | | 20 | 1 | 5.0 | |
| Hashimoto's Thyroiditis | | 19 | 2 | 10.5 | |
| Total Controls | | 600 | 30 | 5.0% | |
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Alegria Flash SSB:
| | Diagnosis | | | | |
| --- | --- | --- | --- | --- | --- |
| | | SLE | SjS | Controls | Totals |
| Alegria Flash SSB | Positive | 36 | 69 | 10 | 115 |
| | Negative | 142 | 65 | 590 | 797 |
| | Total | 178 | 134 | 600 | 912 |
| | SLE | Sensitivity | 20.2% | (95%CI 14.0 – 25.5) | |
| | SjS | Sensitivity | 51.5% | (95%CI 43.1 – 59.8) | |
| | | Specificity | 98.3 | (95%CI 97.0 – 99.1) | |
| SSB Distribution of clinical study samples and antibody positivity rates: | | | |
| --- | --- | --- | --- |
| Diagnostic Group | N | SSA-60 Positive | |
| | | n | (%) |
| Target conditions | | | |
| Systemic lupus erythematosus (SLE) | 178 | 36 | 20.2 |
| Sjögren’s Syndrome (SjS) | 134 | 69 | 51.5 |
| Differential diagnosis controls | | | |
| Systemic sclerosis (SSc) | 124 | 3 | 2.4 |
| Polymyositis (PM) | 50 | 1 | 2.0 |
| Dermatomyositis (DM) | 50 | 2 | 4.0 |
| Mixed Connective Tissue Disease | 20 | 0 | 0 |
| Autoimmune Hepatitis | 30 | 1 | 3.33 |
| Antiphospholipid Syndrome | 20 | 0 | 0 |
| Cancer | 19 | 0 | 0 |
| Celiac Disease | 24 | 0 | 0 |
| Drug Induced Lupus | 5 | 0 | 0 |
| Fibromyalgia | 20 | 0 | 0 |
| Crohn's Disease | 30 | 0 | 0 |
| Ulcerative Colitis | 29 | 0 | 0 |
| HBV | 17 | 0 | 0 |
| HIV | 12 | 0 | 0 |
| HCV | 10 | 0 | 0 |
| HSV | 10 | 0 | 0 |
| Primary Biliary Cholangitis | 30 | 0 | 0 |
| Rheumatoid Arthritis | 20 | 1 | 5.0 |
| Vasculitis (Undifferentiated) | 6 | 0 | 0 |
| Vasculitis (ANCA Associated) | 5 | 0 | 0 |
| Vasculitis (Large/Med Vessel) | 10 | 0 | 0 |
| Atrophic Gastritis | 20 | 0 | 0 |
| Graves’ Disease | 20 | 0 | 0 |
| Hashimoto's Thyroiditis | 19 | 2 | 10.5 |
| Total Controls | 600 | 10 | 1.7% |
# D Clinical Cut-Off:
The assays' cut-offs are 100 Units.
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# E Expected Values/Reference Range:
A negative Alegria Flash SSA-52, Alegria Flash SSA-60, and Alegria Flash SSB test result is expected for apparently healthy individuals. To determine the expected values for the three assays, a panel of 200 apparently healthy blood donors (100 females/100 males, ages 16 to 80 years, mean 51 years of age, median 54 years of age) were tested. The results are as follows:
| Assay | Number of samples positive | Mean concentration (RU) | Range (RU) |
| --- | --- | --- | --- |
| Alegria Flash SSA-52 | 3 (1.5%) | N/A | N/A |
| Alegria Flash SSA-60 | 4 (2.0%) | N/A | N/A |
| Alegria Flash SSB | 1 (0.5%) | 6.0 | 0.0 – 1042.5 |
# VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
# IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
