The FLC Kappa kit is intended for the quantification of Kappa free light chains in human serum from adults with an Enzyme-Linked Immunosorbent Assay (ELISA) procedure. Measurement of free light chains aids in the diagnosis of multiple myeloma and AL amyloidosis. It must be used in conjunction with other laboratory and clinical findings. For In Vitro Diagnostic Use.

The FLC Lambda kit is intended for the quantification of Lambda free light chains in human serum from adults with an Enzyme-Linked Immunosorbent Assay (ELISA) procedure. Measurement of free light chains aids in the diagnosis of multiple myeloma and AL amyloidosis. It must be used in conjunction with other laboratory and clinical findings. For In Vitro Diagnostic Use.

Device Description

The FLC Kappa and FLC Lambda test kits are intended for the quantification of free light chains in human serum from adults with an Enzyme-Linked Immunosorbent Assay (ELISA) procedure utilizing specific antibodies targeting anti-Lambda free light chains. It is carried out in 8 successive steps: Incubation of the previously diluted samples and calibrators, in the wells of the microplate, where specific free light chain antibodies are fixed. Washing of the wells to remove elements that have not been fixed by the anti-free light chain antiserum. Incubation with an anti- light chain antiserum (Kit specific) conjugated to peroxidase. Washing of the wells to remove the excess of antiserum conjugated to peroxidase. Incubation with peroxidase substrate. Stopping of the enzymatic reaction with an acidic solution. Reading of the optical density by absorbance spectrophotometry at 450 nm of the colored product. Calculation of the free light chain concentration of the sample using a calibration curve obtained with calibrators that have been analyzed on the same microplate.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the Sebia FLC Kappa and FLC Lambda kits, based on the provided FDA 510(k) summary.

Note: This document describes an in vitro diagnostic (IVD) device, which measures specific analytes (free light chains) in human serum using a laboratory assay (ELISA). The concepts of "AI assistance," "human readers," "radiologist," and "image" are not relevant to this type of device. Therefore, sections pertaining to these concepts (like MRMC studies) are not applicable and will be marked as such. The "ground truth" for an IVD kit is established through various analytical performance studies demonstrating its accuracy and reliability compared to established methods, and clinical studies correlating its results with disease states.

Acceptance Criteria and Device Performance for Sebia FLC Kappa and FLC Lambda Kits

1. Table of Acceptance Criteria & Reported Device Performance

The FDA 510(k) summary does not explicitly present a "table of acceptance criteria" in the format of pass/fail thresholds. Instead, it details various performance studies and their results, which implicitly form the basis for acceptance. The reported device performance is indicated by the successful execution of these studies and the obtained values meeting recognized clinical laboratory standards (e.g., CLSI guidelines).

General Performance Categories and Key Findings:

Performance Category	Acceptance Criteria (Implicit from CLSI Guidelines/Industry Norms)	Reported Device Performance (Key Findings)
Precision (Reproducibility)	Low Coefficient of Variation (CV%) across various conditions (within-run, between-runs, within-day, between-days, operators, lots, sites). Generally, CV% should be within acceptable limits for diagnostic assays.	FLC Kappa: Total %CVs for single-site, multi-operator, multi-lot, and multi-site reproducibility studies were generally below 15% across various sample concentrations, which is typical for acceptable immunoassay precision. FLC Lambda: Similar to Kappa, total %CVs across various reproducibility studies were generally below 15%, demonstrating acceptable precision. (e.g., Multi-site total reproducibility: 11.4% to 15.2%)
Linearity/Assay Range	Demonstrated linearity across the claimed analytical measuring range, with slopes near 1 and intercepts near 0 for linear regression.	FLC Kappa: Linear between 4.5 mg/L and 76.2 mg/L. Three panels showed linear regressions with slopes close to 1 (e.g., Y=1.009x -0.2967, Y=1.016x -0.2014, Y=0.9332x + 0.4952) and small Y-intercepts. FLC Lambda: Linear between 3.8 mg/L and 66.8 mg/L. Three panels showed linear regressions with slopes close to 1 (e.g., Y=1.046x -1.537, Y=0.9561x -0.62, Y=0.9611x -0.31) and small Y-intercepts.
Limits (LOB, LOD, LOQ)	Limits of Blank (LOB), Detection (LOD), and Quantitation (LOQ) must be established and clinically relevant. LOQ typically related to imprecision (e.g., <20% CV).	FLC Kappa: LOB = 0 mg/L, LOD = 0.8 mg/L, LOQ = 4.5 mg/L. FLC Lambda: LOB = 0 mg/L, LOD = 1.1 mg/L, LOQ = 3.8 mg/L. LOQ defined as lowest concentration with <20% within-laboratory imprecision.
Hook Effect	No significant Hook effect (antigen excess) within the expected clinical range.	No interference driven by the Hook effect was observed.
Analytical Specificity (Interferences)	No significant interference from common biological substances or drugs at clinically relevant concentrations.	No interference detected from conjugated/unconjugated bilirubin, total protein, hemoglobin, triglycerides, rheumatoid factor, or specified drugs (melphalan, dexamethasone, daratumumab, bortezomib, lenalidomide, pomalidomide, carfilzomib, isatuximab) at tested levels.
Stability	Kits and controls must maintain stated performance over their claimed shelf-life and in-use periods.	FLC Kappa & Lambda Kits: Shelf life: 9 months at 2-8°C. In-use: 5 uses within 1 month and 15 cumulative hours maximum at room temperature (19-25°C). All results met stability acceptance criteria.
Method Comparison (Quantitative)	Demonstrated agreement with a legally marketed predicate device (Siemens BN™ II Freelite® assays), often assessed by regression analysis (slope, intercept, R2).	FLC Kappa (vs. predicate): N=216, Sample Range 1.0-1947.0 mg/L. Slope: 0.557 (95% CI: 0.5125-0.6021), Y-Intercept: 0.912 (95% CI: 0.752-1.072), R2: 0.917. FLC Lambda (vs. predicate): N=221, Sample Range 1.6-860.4 mg/L. Slope: 0.608 (95% CI: 0.5224-0.6937), Y-Intercept: 2.243 (95% CI: 1.149-3.337), R2: 0.749. Note: Slopes deviate significantly from 1, indicating a proportional bias. This is common when comparing different assay methods and platforms, and is acceptable as long as the correlation is strong and clinical utility is demonstrated.
Method Comparison (Qualitative - Ratio)	Acceptable Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) with the predicate for the KFLC/LFLC ratio.	Kappa FLC / Lambda FLC Ratio (vs. predicate): PPA = 82.3%, NPA = 89.3%. (N=216 serum samples).
Clinical Performance (Sensitivity/Specificity)	Acceptable clinical sensitivity and specificity for indicated conditions (multiple myeloma, AL amyloidosis), demonstrated by correlation with clinical diagnosis.	Multiple Myeloma (N=366): Clinical sensitivity: 96.6% (95% CI: 94.0%-99.3%), Clinical specificity: 85.1% (95% CI: 79.4%-89.5%).AL Amyloidosis (N=333): Clinical sensitivity: 91.0% (95% CI: 86.3%-95.7%), Clinical specificity: 85.1% (95% CI: 79.4%-89.5%).
Reference Range	Established reference intervals for healthy populations.	US-population (N=238 healthy adults): Kappa FLC: 6.4 - 17.4 mg/L. Lambda FLC: 8.4 - 21.8 mg/L. Kappa FLC / Lambda FLC ratio: 0.46 - 1.51.

2. Sample Sizes and Data Provenance

Precision (Reproducibility):
- Single-site: 6 samples per kit (4 serum, 2 controls), 3 replicates/sample, 20 days, 2 runs/day = 120 results per sample.
- Multi-operator: 6 samples per kit (4 serum, 2 controls), 5 replicates/sample, 5 days, 3 operators = 75 results per sample.
- Multi-lot: 6 samples per kit (4 serum, 2 controls), 5 replicates/sample, 5 days, 3 lots = 75 results per sample.
- Multi-site: 6 samples per kit (4 serum, 2 controls), 5 replicates/sample, 5 days, 3 sites (3 operators).
Linearity: 3 patient sample pools for each FLC assay (Kappa, Lambda). Number of individual samples/aliquots per pool not specified, but typically involves serial dilutions.
LOD/LOQ: 5 low-concentration samples, tested in 2 runs of 4 replicates over 7 days using 2 reagent lots (56 replicates/sample/lot).
Method Comparison (Quantitative): 216 serum samples (Kappa), 221 serum samples (Lambda).
Method Comparison (Qualitative - Ratio): 216 serum samples.
Reference Range: 238 apparently healthy adults (US-population).
Clinical Study: 510 samples total (177 Multiple Myeloma, 144 AL Amyloidosis, 189 non-myeloma/non-amyloidosis subjects with various clinical conditions).
Data Provenance: The document explicitly states the reference range study was conducted on a "US-population." The clinical study involved samples from various clinical conditions, implying real-world patient data, likely retrospective given the disease categories. The other analytical studies were performed in a controlled laboratory setting. The exact countries of origin for all samples beyond the US reference range study are not specified, but the manufacturing site is in France, suggesting international collaboration or testing. All studies appear to be prospective in their design for generating the performance data for the submission.

3. Number of Experts & Qualifications for Ground Truth

This is an IVD device, not an AI image analysis device. As such, there are no "experts" in the sense of radiologists providing interpretations for AI model ground truth. The "ground truth" for the performance studies is established by:

Reference Methods: Comparison studies use a predicate device (The Binding Site Freelite® Human Kappa/Lambda Free kits on Siemens BN™ II) as a reference.
Established CLSI Guidelines: All analytical studies (precision, linearity, limits, interference) follow rigorous Clinical and Laboratory Standards Institute (CLSI) guidelines (e.g., EP05-A3, EP06-ed2, EP17-A2, EP7-A2, EP09-A3, EP28-A3c). Compliance with these guidelines, developed by consensus among clinical laboratory experts, forms the basis for acceptable performance.
Clinical Diagnosis: For the clinical study, the "ground truth" for multiple myeloma and AL amyloidosis was the clinical diagnosis of the patients. This diagnosis would be established by medical specialists (e.g., hematologists, oncologists) based on a comprehensive evaluation of laboratory findings, biopsy results, imaging, and clinical presentation. No specific number of such adjudicating clinicians is provided.

4. Adjudication Method for the Test Set

Not applicable in the context of an IVD device measuring analytes. The ground truth for analytical performance is adherence to validated laboratory procedures and statistical analysis per CLSI guidelines, and for clinical performance, it's the established clinical diagnosis for the patient samples.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

Not applicable. This is an in vitro diagnostic assay, not an AI-assisted diagnostic imaging device that involves human readers interpreting images with or without AI.

6. Standalone Performance

Yes, the entire performance data provided (precision, linearity, limits, interference, stability, quantitative and qualitative comparisons) represents the standalone performance of the Sebia FLC Kappa and FLC Lambda ELISA kits. The results are generated directly by the assay without human interpretation of complex outputs like images. The only "human-in-the-loop" aspect is the performance of the laboratory testing, which is standardized by the kit's instructions for use and quality control.

7. Type of Ground Truth Used

Analytical Performance: Ground truth derived from established laboratory reference methods (e.g., predicate device for comparison), consensus guidelines (CLSI), and defined metrological properties (e.g., purified standards for linearity, known low-concentration samples for LOD/LOQ).
Clinical Performance: "Ground truth" for the clinical study was the clinical diagnosis (Multiple Myeloma and AL Amyloidosis) of the patients from whom the serum samples were collected. This is based on comprehensive clinical and laboratory findings, not a single test.

8. Sample Size for the Training Set

Not applicable. This is not an AI/machine learning device that requires a training set. This is a traditional IVD ELISA assay.

9. How the Ground Truth for the Training Set Was Established

Not applicable, as there is no "training set" for this type of IVD device.

Summary

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health and Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

November 18, 2022

Sebia, Inc. Karen Anderson Director of Regulatory 1705 Corporate Drive, Suite 400 Norcross, Georgia 30093

Re: K210623

Trade/Device Name: FLC Kappa, FLC Lambda Regulation Number: 21 CFR 866.5550 Regulation Name: Immunoglobulin (Light Chain Specific) Immunological Test System Regulatory Class: Class II Product Code: DFH, DEH Dated: August 2, 2022 Received: August 4, 2022

Dear Karen Anderson:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

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Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Ying Mao -S

Ying Mao, Ph.D. Branch Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K210623

Device Name FLC Kappa FLC Lambda

Indications for Use (Describe)

Type of Use (Select one or both, as applicable)
☑ Prescription Use (Part 21 CFR 801 Subpart D)	☐ Over-The-Counter Use (21 CFR 801 Subpart C)

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510K SUMMARY (Summary of Safety and Effectiveness)

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

Submitter Name	Sebia, Inc.
Address	1705 Corporate Drive Suite 400Norcross, Georgia 30093, USA
Contact	Karen Anderson, Dir of Regulatory, Sebia Inc.Phone: 1-800-835-6497Fax: 770-446-8511Email: karen.anderson@sebia-usa.comMatthew C Wagner, Scientific Affairs SpecialistPhone 1-800-835-6497, 3752Fax 770-446-8511Email: Matthew.Wagner@sebia-usa.com
Date Prepared	October 20, 2022
Manufacturing	SebiaParc Technologique Léonard de VinciRue Léonard de Vinci,CP 8010 LISSES, 91008 EVRY CedexFRANCEPhone: (33) 1 69 89 80 80Fax: (33) 1 69 89 78 78
Product Name	FLC KappaFLC Lambda
Common Name	Light Chain immunological test system
Product Regulation No.	21CFR sec. 866.5550 - Immunoglobulin (light chainspecific) immunological test system21 CFR sec. 862.1660- Quality Control Material(assayed and unassayed)
Product Codes	DFH, Kappa antigen, antiserum, controlDEH, Lambda, antigen, antiserum, control
Device classification and PanelClassification	Class II, Immunology (82)
Establishment Registration No.	8023024

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1. DEVICE DESCRIPTIONS

It is carried out in 8 successive steps:

Incubation of the previously diluted samples and calibrators, in the wells of the । microplate, where specific free light chain antibodies are fixed.
Washing of the wells to remove elements that have not been fixed by the anti-free light chain antiserum.
Incubation with an anti- light chain antiserum (Kit specific) conjugated to peroxidase. -
Washing of the wells to remove the excess of antiserum conjugated to peroxidase. -
Incubation with peroxidase substrate. -
Stopping of the enzymatic reaction with an acidic solution. -
-Reading of the optical density by absorbance spectrophotometry at 450 nm of the colored product.
-Calculation of the free light chain concentration of the sample using a calibration curve obtained with calibrators that have been analyzed on the same microplate.

2. REAGENTS

REAGENTS AND MATERIALS SUPPLIED IN THE FLC KAPPA AND FLC LAMBDA KITS

	PN 5102	PN 5103
ITEMS	FLC Kappa Kit	FLC Lambda Kit
Microplate Kappa 1 plate with 12 segments, 8 wells each	Microplate Kappa 1 plate with 12 segments, 8 wells each	Microplate Lambda 1 plate with 12 segments, 8 wells each
Dilution buffer (ready to use)	1 vial, 100 mL	1 vial, 100 mL
Wash solution (stock solution)	1 vial, 100 mL	1 vial, 100 mL
Calibrators (ready to use)	5 vials, 0.6 mL eachKappa Calibrators	5 vials, 0.6 mL eachLambda Calibrators
PER antiserum (ready to use)	1 vial, 12 mLAnti-Kappa - PER antiserum (ready to use)	1 vial, 12 mLAnti-Lambda - PER antiserum (ready to use)
Substrate	1 vial, 12 mLSubstrate Kappa (ready to use)	1 vial, 12 mLSubstrate Lambda (ready to use)
Stop solution (ready to use)	1 vial, 12 mL	1 vial, 12 mL

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REAGENTS/SUPPLIES REQUIRED BUT NOT SUPPLIED IN THE KITS

SUPPLIES	SEBIA PRODUCT NUMBER
Densitometer for microplate reading byabsorbance spectrophotometry at 450 nm.
NON COATED ELISA PLATES (10), SEBIA,microplates with scored wells in order tocomplete segments when the forecastednumber of samples per segment is fewer than8, and frameto store the segments that have not beenused.	PN 5303
Absorbent paper for removal the excess ofwash solution from the wells of the microplate.
Multichannel pipette.
FLC CONTROL LEVEL 1	PN 5112
FLC CONTROL LEVEL 2	PN 5113

3. INDICATIONS FOR USE

FLC Kappa and FLC Lambda kits

For In Vitro Diagnostic Use only.

Special conditions for use statements: For prescription use only.

Warning: The result of the FLC Kappa and FLC Lambda in a given specimen determined with assays and/or instrument platforms from different manufacturers can vary due to differences in assay methods and reagent

specificity. The results reported by the laboratory to the physician must include the identity of the assay used. Values obtained with different assay methods cannot be used interchangeably.

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SUBSTANTIAL EQUIVALENCE INFORMATION

Predicate Device Name	Predicate Device510(k) number	Regulation No.
The Binding Site Freelite®Human Kappa Free Kit for use onthe Siemens BN™ II	K031016	866.5550
The Binding Site Freelite®Human Lambda Free Kit for useon the Siemens BN™ II	K031016	866.5550

4. COMPARISON WITH PREDICATE DEVICE

Kit Similarities (Table A).

Similarities
	Candidate Device	Predicate
Table A	FLC KappaFLC Lambda	The Binding Site Freelite® Human Kappa Freeand Freelite® Human Lambda Free kits for useon the Siemens BN TM II (K031016)
Analyte	Kappa: Kappa FLCLambda: Lambda FLC	Same
Measurement	Quantitative	Same

Kit Differences (Table B)

	Differences
Table B	Candidate Device	Predicate Device
Indications for Use	The FLC Kappa kit is intended for thequantification of Kappa free lightchains in human serum from adultswith an Enzyme-LinkedImmunosorbent Assay (ELISA)procedure. Measurement of free lightchains aids in the diagnosis of multiplemyeloma and AL amyloidosis. It mustbe used in conjunction with otherlaboratory and clinical findings.For In Vitro Diagnostic Use only.	Kappa: This kit is intended for thequantitation of kappa free light chains inserum and urine on the Siemens BN™ II.Measurement of free light chains aids in thediagnosis and monitoring of multiplemyeloma, lymphocytic neoplasms,Waldenstrom's macroglobulinemia, ALamyloidosis, light chain deposition diseaseand connective tissue diseases such assystemic lupus erythematosus inconjunction with other laboratory and clinicalfindings.

	The FLC Lambda kit is intended for thequantification of Lambda free lightchains in human serum from adultswith an Enzyme-LinkedImmunosorbent Assay (ELISA)procedure. Measurement of free lightchains aids in the diagnosis of multiplemyeloma and AL amyloidosis. It mustbe used in conjunction with otherlaboratory and clinical findings.For In Vitro Diagnostic Use only.	Lambda: This kit is intended for thequantitation of lambda free light chains inserum and urine on the Siemens BN™ IIMeasurement of free light chains aids in thediagnosis and monitoring of multiplemyeloma, lymphocytic neoplasms,Waldenstrom's macroglobulinemia, ALamyloidosis, light chain deposition diseaseand connective tissue diseases such assystemic lupus erythematosus inconjunction with other laboratory and clinicalfindings.
SpecimenType	Human Serum	Human Serum, Human Urine
DetectionMethod	Enzyme-Linked ImmunosorbentAssay (ELISA)	Nephelometric
DetectionAntibody	Kappa : Sandwich ELISA withpolyclonal rabbit anti- humankappa free light chains coatedon the well of the microplate(capture antibody) andpolyclonal rabbit anti-humankappa light chain conjugated tohorseradish peroxidase (HRP)(detection antibody)Lambda : Sandwich ELISA withpolyclonal rabbit anti-human lambdafree light chain coated on the well ofthe microplate (capture antibody) andpolyclonal rabbit anti-human lambdalight chain conjugated to horseradishperoxidase (HRP) (detection antibody)	Kappa: Polyclonal sheep anti-human kappaantibody coated with latex particlesLambda: Polyclonal sheep anti-humanLambda antibody coated with latex particles
AnalyticalMeasuringranges	KappaStandard dilution (1/1000)4.5 to 76.2 mg/L(dilution scheme 1/250 to 1/100 000)LambdaStandard dilution (1/1000)3.8 to 66.8 mg/L(dilution scheme 1/250 to 1/100 000)	KappaStandard dilution (1/100)5.9 to 190 mg/L(dilution scheme 1/5 to 1/8000)LambdaStandard dilution (1/100)5.0 to 160 mg/L(dilution scheme 1/5 to 1/8000)
ReferenceInterval	Kappa: 6.4 to 17.4 mg/L.Lambda: 8.4 to 21.8 mg/L.Ratio: 0.46 to 1.51	Kappa: 3.30 to 19.40 mg/LLambda: 5.71 to 26.30 mg/LRatio: 0.26-1.65
Similarities
Item	Candidate	Predicate
Reference Material	Internal preparation	Reference Same

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Calibrators:

Differences
Item	Candidate	Predicate
Number of levels	5	One

5. PERFORMANCE DATA

a) Precision and Reproducibility

The precision and FLC Kappa and FLC Lambda assays were evaluated according to the CLSI EP05-A3 guideline.

Single-site reproducibility

Six different samples were tested using the FLC Kappa and FLC Lambda kits. The analyzed samples included 4 serum samples S1 to S4) and 2 controls (samples C1 and C2). Each day, for 20 days, a unique operator analyzed the same samples (3 replicates / sample) on a microplate (2 runs with minimum 2 hours between the 2 runs) with 1 reagent lot (same microplate design each day), yielding a total of 120 results per sample.

The single-site reproducibility study is summarized in the following table including within-run, between runs, within-day, between-days and total reproducibility precision estimates (SD and %CV) for the free light chain concentrations (in mg/L).

Sample	Mean(mg/L)	Within-run		Between-runs		Within-day		Between-days		Total(*)reproducibility(Within-laboratoryprecision)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	39.2	1.47	3.8%	4.02	10.2%	4.28	10.9%	1.29	3.3%	4.47	11.4%
S2	6.3	0.41	6.6%	0.74	11.9%	0.85	13.6%	0.36	5.8%	0.92	14.8%
S3	14.4	0.54	3.7%	1.30	9.0%	1.40	9.7%	0.81	5.6%	1.62	11.2%
S4	62.0	2.89	4.7%	6.03	9.7%	6.69	10.8%	1.77	2.9%	6.92	11.2%
C1	11.5	0.67	5.8%	1.09	9.5%	1.28	11.2%	0.00	0.0%	1.28	11.2%
C2	42.2	1.64	3.9%	4.00	9.5%	4.32	10.2%	0.66	1.6%	4.37	10.4%

FLC Kappa

(*) Total reproducibility includes within-run, between-runs, within-day and between-days.

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FLC Lambda

Sample	Mean(mg/L)	Within-run		Between-runs		Within-day		Between-days		Totalreproducibility(Within-laboratoryprecision)(*)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	33.7	1.55	4.6%	2.67	7.9%	3.09	9.2%	1.61	4.8%	3.48	10.3%
S2	13.2	0.55	4.2%	0.96	7.3%	1.11	8.4%	0.63	4.8%	1.28	9.7%
S3	20.0	0.83	4.1%	1.25	6.2%	1.50	7.5%	0.74	3.7%	1.67	8.3%
S4	79.5	5.13	6.5%	5.15	6.5%	7.27	9.1%	6.91	8.7%	10.03	12.6%
C1	19.7	1.33	6.8%	1.40	7.1%	1.93	9.8%	0.00	0.0%	1.93	9.8%
C2	37.1	1.66	4.5%	2.76	7.5%	3.22	8.7%	1.55	4.2%	3.57	9.6%

(*) Total reproducibility includes within-run, between-runs, within-day and between-days.

Multi-operator reproducibility

Six different samples were tested using the FLC Kappa and FLC Lambda kits. The analyzed samples included 4 serum samples (samples S1 to S4) and 2 controls (samples C1 and C2). Each day, for 5 days, 3 operators analyzed the same samples (5 replicates / sample) on a microplate, with 1 reagent lot (same microplate design each day), yielding a total of 75 results per sample. The multi-operator reproducibility study is summarized in the following within-day, between-days, within-operator, between-operators and total reproducibility precision estimates (SD and %CV) for the free light chain concentrations (in mg/L).

Sample	Mean(mg/L)	Within-day		Between-days		Within-operator		Between-operators		Totalreproducibility (*)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	44.1	2.49	5.7%	2.05	4.7%	3.23	7.3%	0.50	1.1%	3.27	7.4%
S2	6.8	0.46	6.8%	0.63	9.2%	0.78	11.4%	0.00	0.0%	0.78	11.4%
S3	15.7	0.72	4.6%	1.30	8.3%	1.49	9.5%	0.00	0.0%	1.49	9.5%
S4	67.9	3.52	5.2%	5.58	8.2%	6.60	9.7%	0.00	0.0%	6.60	9.7%
C1	12.7	1.25	9.9%	0.93	7.3%	1.56	12.3%	0.50	3.9%	1.64	12.9%
C2	44.8	1.95	4.3%	2.94	6.6%	3.53	7.9%	0.00	0.0%	3.53	7.9%

FLC Kappa

(*) Total reproducibility includes within-day, between-days, within-operator and between-operators.

FLC Lambda

Sample	Mean(mg/L)	Within-day		Between-days		Within-operator		Between-operators		Totalreproducibility (*)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	35.4	2.70	7.6%	2.87	8.1%	3.94	11.1%	0.00	0.0%	3.94	11.1%
S2	13.4	0.87	6.5%	0.83	6.2%	1.20	9.0%	0.00	0.0%	1.20	9.0%
S3	21.1	0.98	4.6%	1.49	7.1%	1.79	8.5%	0.00	0.0%	1.79	8.5%
S4	81.5	5.88	7.2%	5.58	6.8%	8.11	10.0%	3.99	4.9%	9.03	11.1%
C1	20.7	1.59	7.7%	1.41	6.8%	2.13	10.3%	0.00	0.0%	2.13	10.3%
C2	37.1	2.09	5.6%	2.81	7.6%	3.51	9.4%	1.93	5.2%	4.00	10.8%

(*) Total reproducibility includes within-day, between-days, within-operator and between-operators.

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Multi-lot reproducibility

Six different samples were tested using the FLC Kappa and FLC Lambda kits. The analyzed samples included 4 serum samples $1 to S4), and 2 controls (samples C1 and C2), Each day, for 5 days, 1 operator analyzed the same samples (5 replicates / sample) on a microplate, with 3 reagent lots (1 microplate per reagent lot, same microplate design each day), yielding a total of 75 results per sample. The multi-lot reproducibility study is summarized in the following table including between-days, within day, between-lots, within-lot and total reproducibility precision estimates (SD and %CV) for the free light chain concentrations (in mg/L).

FLC Kappa

Sample	Mean(mg/L)	Between-days		Within-day		Between-lots		Within-lot		Total reproducibility(*)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	38.7	1.72	4.4%	2.00	5.2%	0.76	2.0%	2.64	6.8%	2.75	7.1%
S2	6.8	0.50	7.4%	0.36	5.3%	0.17	2.6%	0.62	9.1%	0.64	9.4%
S3	15.3	0.55	3.6%	0.50	3.3%	0.40	2.6%	0.74	4.9%	0.84	5.5%
S4	62.2	1.69	2.7%	3.59	5.8%	1.65	2.6%	3.96	6.4%	4.29	6.9%
C1	12.1	0.54	4.4%	0.86	7.1%	0.00	0.0%	1.01	8.3%	1.01	8.3%
C2	42.8	1.71	4.0%	1.96	4.6%	1.44	3.4%	2.60	6.1%	2.97	6.9%

(*) Total reproducibility includes between-days, within-day, between-lots and within-lot.

FLC Lambda

Sample	Mean(mg/L)	Between-days		Within-day		Between-lots		Within-lot		Totalreproducibility (*)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	33.7	3.12	9.3%	2.91	8.6%	0.70	2.1%	4.27	12.7%	4.33	12.9%
S2	13.8	1.20	8.7%	1.14	8.3%	1.40	10.1%	1.66	12.0%	2.17	15.7%
S3	20.8	1.98	9.5%	0.99	4.8%	2.20	10.6%	2.21	10.7%	3.12	15.0%
S4	71.4	7.63	10.7%	5.71	8.0%	0.00	0.0%	9.53	13.4%	9.53	13.4%
C1	20.9	1.51	7.2%	1.70	8.2%	2.32	11.1%	2.27	10.9%	3.25	15.6%
C2	38.5	3.29	8.5%	1.84	4.8%	1.66	4.3%	3.77	9.8%	4.12	10.7%

(*) Total reproducibility includes between-days, within-day, between-lots and within-lot.

Multi-site reproducibility

Six different samples were tested using the FLC Kappa and the FLC Lambda kits. The analyzed samples included 4 serum samples (samples S1 to S4) and 2 controls (samples C1 and C2). Each day, for 5 days, 1 operator analyzed the same samples (1 dilution 1/1000, 5 replicates / sample) on a microplate with 1 reagent lot (same microplate design each day). The same protocol was followed by 2 other operators in 2 other laboratories with the same reagent lot.

The multi-sites reproducibility study is summarized in the following within-day, between-days, within-site, between-sites and total reproducibility precision estimates (SD and %CV) for the free light chain concentrations (in mg/L).

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FLC Kappa

Sample	Mean(mg/L)	Between-days		Within-day		Between-sites		Within-site		Total reproducibility(*)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	6.2	0.39	6.3%	0.45	7.2%	0.00	0.0%	0.60	9.6%	0.60	9.6%
S2	15.0	1.48	9.9%	0.87	5.8%	0.00	0.0%	1.71	11.4%	1.71	11.4%
S3	32.5	2.76	8.5%	1.29	4.0%	1.48	4.5%	3.05	9.4%	3.38	10.4%
S4	71.9	5.78	8.0%	4.23	5.9%	2.86	4.0%	7.16	9.9%	7.71	10.7%
C1	13.8	0.70	5.0%	1.08	7.9%	1.69	12.2%	1.29	9.3%	2.12	15.4%
C2	57.2	2.68	4.7%	2.67	4.7%	3.79	6.6%	3.78	6.6%	5.36	9.4%

(*) Total reproducibility includes between-days, within-day, between-sites and within-site.

FLC Lambda

Sample	Mean (mg/L)	Between-days		Within-day		Between-sites		Within-site		Total reproducibility (*)
		SD	CV	SD	CV	SD	CV	SD	CV	SD	CV
S1	11,4	0,91	8,0%	0,76	6,7%	0,51	4,5%	1,19	10,5%	1,30	11,4%
S2	20,7	1,62	7,8%	1,51	7,3%	1,12	5,4%	2,22	10,7%	2,48	12,0%
S3	31,3	3,39	10,8%	2,12	6,8%	1,87	6,0%	4,00	12,8%	4,42	14,1%
S4	68,8	6,13	8,9%	4,44	6,5%	4,81	7,0%	7,57	11,0%	8,97	13,0%
C1	24,2	2,27	9,4%	1,62	6,7%	2,40	9,9%	2,78	11,5%	3,68	15,2%
C2	50,1	5,81	11,6%	3,87	7,7%	2,83	5,7%	6,98	13,9%	7,53	15,1%

(*) Total reproducibility includes between-days, within-day, between-sites and within-site.

b) Linearity/assay

The linearity of the FLC Kappa and FLC Lambda kits was evaluated in a study based on the Clinical and Laboratory Standards Institute (CLSI - USA) EP06-ed2: 2020 guideline "Evaluation of the Linearity of Quantitative Measurement Procedures".

The results for concentration (mg/L) of Kappa and Lambda free light chains were analyzed using statistical tools recommended by CLSI.

Three linearity panels at 1/1000 dilution were performed based on 3 different patient sample pools.

FLC Assay	RangeTested(mg/L)	LinearRegression	95% CI Slope	95%CIY-Intercept	Claimedlinearrange
Kappa study N°1	[4.4; 81.7]	Y=1.009x -0.2967	[0.9534; 1.065]	[-0.88; 0.29]
Kappa study N°2	[4.5; 79.9]	Y=1.016x -0.2014	[0.967; 1.065]	[-0.72; 0.32]	[4.5mg/L;76.2mg/L]
Kappa study N°3	[3.5; 76.2]	$Y=0.9332x + 0.4952$	[0.8635; 1,003]	[-0.11; 1.10]
Lambda study N°1	[3.8; 74.1]	Y=1.046x -1.537	[0.9389; 1.153]	[-2.52; -0.55]
Lambda study N°2	[3.3; 77.7]	Y=0.9561x -0.62	[0.9299; 0.9823]	[-0.84; -0.40]	[3.8mg/L;66.8mg/L]
Lambda study N°3	[3.5; 66.8]	Y=0.9611x -0.31	[0.8948; 1.027]	[-0.87; 0.25]

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The highest of the lower limit and the lowest of the higher limit found in the 3 linearity panels were taken.

The linearity of FLC Kappa kit at initial dilution 1/1000 is demonstrated between 4.5 mg/L and 76.2 ma/L.

The linearity of FLC Lambda kit at initial dilution 1/1000 is demonstrated between 3.8 mg/L and 66.8 mq/L.

c) Limit of Blank (LOB) / Limit of Detection (LOD) / Limit of Quantitation (LOQ)

The determination of the limit of detection (LOD) and of the limit of quantitation (LOQ) of the FLC Kappa kit was evaluated in a study based on the Clinical and Laboratory Standards Institute (CLSI - USA) EP17-A2 guideline "Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition".

Due to the blank removal, the limit of blank (LOB) is considered as being 0 mg/L.

The LOD was determined by assaying 5 samples with low Kappa free light chains concentration: they were tested in two runs of four replicates over the course of seven days using two reagent lots (a total of 56 replicates per sample for each lot). The precision profile analysis was used to calculate the LOD. Since LOD values for the 2 lots are below the calibrator 1 values, the LOD value is considered as equal to the LOQ value.

The LOQ was determined by assaying 5 samples with low Kappa free light chains concentration: they were tested in two runs of four replicates over the course of seven days using two reagent lots (a total of 56 replicates per sample for each lot). The LoQ was defined to be the lowest concentration level that meets the within-laboratory imprecision of < 20% for each lot. Since LOQ values for the 2 lots are below the linearity study range, the LOQ value is considered to be equal to the concentration of the lower limit sample with the highest value among the 3 linearity panels.

The claimed LoB/LoQ for the FLC Kappa and FLC Lambda kits are as follows:

Kit	LOB	LOD	LOQ
FLC Kappa	0 mg/L	0.8 mg/L	4.5 mg/L
FLC Lambda	0 mg/L	1.1 mg/L	3.8 mg/L

d) Hook Effect (Antigen Excess)

No interference driven by the Hook effect was observed with the FLC Kappa and FLC Lambda kits.

e) Traceability

In the absence of an international reference standard, the calibration of the assay is traceable to an internally assigned master calibrator.

f) Analytical specificity

Biological Interferences:

The common interfering factors with the FLC Kappa and FLC Lambda kits (conjugated bilirubin, unconjugated bilirubin, total protein, hemoglobin, triglycerides and rheumatoid factor) were evaluated in studies based on the Clinical Laboratory Standards Institute (CLSI - USA) EP7-A2 guideline "Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition" and the

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Clinical Laboratory Standards Institute (CLSI -USA) EP07, 3rd Edition quideline "Interference Testing in Clinical Chemistry". The results are summarized below.

No interference with the FLC Kappa and FLC Lambda kits was detected due to the serum sample's high concentration of the following interfering factors tested at levels equal to the concentrations listed below:

Endogenous (Biological)	FLC Kappa	FLC Lambda
Conjugated bilirubin	66.6 mg/dL	66.6 mg/dL
Unconjugated bilirubin	40 mg/dL	40 mg/dL
Total Protein	150 g/L	150 g/L
Hemoglobin	10 g/L	10 g/L
Triglycerides	20 g/L	20 g/L
Rheumatoid Factor	2000 IU/mL	250 IU/mL

Drug Interferences:

The common interfering factors with the FLC Kappa and FLC Lambda kits (melphalan, dexamethazon daratumumab, bortezomib, lenalidomide, carfilzomib, isatuximab) were evaluated in studies based on the Clinical Laboratory Standards Institute (CLSI - USA) EP7-A2 guideline "Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition" and Clinical Laboratory Standards Institute (CLSI - USA) EP07, 3rd Edition guideline "Interference Testing in Clinical Chemistry". The results are summarized below.

No interference with the FLC Lambda kit was detected due to the serum sample's high concentration of the following interfering factors tested at levels equal to the concentrations listed below:

Drug	FLC Kappa	FLC Lambda
Melphalan	4 mg/L	4 mg/L
Dexamethazon	12 mg/L	12 mg/L
Daratumumab	1 g/L	1 g/L
Bortezomib	2 mg/L	2 mg/L
Lenalidomide	4 mg/L	4 mg/L
Pomalidomide	1 mg/L	1 mg/L
Carfilzomib	1 mg/L	1 mg/L
Isatuximab	1 g/L	1 g/L

g) Stability

Shelf-life, in-use studies were conducted using the FLC Kappa and FLC Lambda kits and FLC controls Level 1 and Level 2. All results met stability acceptance criteria and product stability claims as listed in the tables below:

Stability of Kit (FLC Kappa and FLC Lambda)

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Kit	Shelf Life	In-use
FLC Kappa	9 months at 2 - 8 °C	5 uses within 1 month and 15 cumulative hours maximum at room temperature (19-25°C)
FLC Lambda	9 months at 2 - 8 °C	5 uses within 1 month and 15 cumulative hours maximum at room temperature (19-25°C)

h) Expected values/ Reference range

The reference range intervals for the FLC Kappa and FLC Lambda kits were performed following the CLSI EP28-A3c, "Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory'.

The studies were performed using a total of 238 apparently healthy adults (US-population). For Kappa free light chain concentration and Lambda free light chain concentration, the reference intervals were calculated nonparametrically and represent the central 95% range of the population, with a 95% confidence level:

Kappa free light chain concentration: 6.4 - 17.4 mg/L Lambda free light chain concentration: 8.4 - 21.8 mg/L

For the [Kappa free light chain] / [Lambda free light chain] ratio, the reference interval represents 100% of the samples:

[Kappa free light chain] / [Lambda free light chain] ratio: 0.46 - 1.51

The Package insert states:" It is recommended each laboratory establish its own reference values."

i) Comparison studies

Serum samples were tested with the Sebia FLC Kappa and FLC Lambda kits and results were compared to the predicate device. The 222 serum samples were tested spanning the dynamic range of one or both assays used in this study performed in the USA.

Quantitative comparison

The FLC Kappa and FLC Lambda kits was evaluated in a study based on the Clinical and Laboratory Standards Institute (CLSI - USA) EP09-A3 quideline "Measurement Procedure Comparison and Bias Estimation Usinq Patient Samples; Approved Guideline - Third Edition". The results for Kappa and Lambda free light chain concentrations (mg/L) were analyzed using statistical tools recommended by CLSI.

The levels of Kappa free light chains were measured in 216 serum samples with normal and abnormal Kappa free light chain concentration. The measured values for Kappa free light chain concentrations from both procedures were analyzed by a weighted Deming regression analysis. The results of the regression analysis are tabulated below (y = FLC Kappa):

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Kit	N	Sample RangeSebia FLC mg/L	Slope	95% CI(Slope)	Y-Intercept	95% CI(Y-Intercept)	R2
FLC Kappa	216	1.0-1947.0	0.557	0.5125-0.6021	0.912	0.752-1.072	0.917

The levels of Lambda free light chains were measured in 221 serum samples with normal and abnormal Lambda free light chain concentration. The measured values for Lambda free light chain concentrations from both procedures were analyzed by a weighted Deming regression analysis. The results of the regression analysis are tabulated below (y = FLC Lambda):

Kit	N	Sample RangeSebia FLC mg/L	Slope	95% CI(Slope)	Y-Intercept	95% CI(Y-Intercept)	R2
FLC Lambda	221	1.6-860.4	0.608	0.5224-0.6937	2.243	1.149-3.337	0.749

Qualitative comparison

[Kappa free light chain] / [Lambda free light chain] ratio ([Kappa FLC] / [Lambda FLC] ratio)

The levels of Kappa free light chains and Lambda free light chains were measured in 216 serum samples using the FLC Kappa and the FLC Lambda kits and a commercially available nephelometric technique (reference), for the calculation of the [Kappa FLC] / [Lambda FLC] ratio of each sample.

The positive percent agreement (PPA) and negative percent agreement (NPA) of the FLC Kappa and FLC Lambda kits compared to the commercially available nephelometric technique have been calculated. The results are tabulated below:

	Range of valuesFLC Kappa / Lambda Ratio	Positive PercentAgreement (%)	Negative PercentAgreement (%)
Kappa free light chain / Lambdafree light chain Ratio	0,00 - 671,38	82,3	89,3

i) Clinical Study

Diagnosis of multiple myeloma and AL amyloidosis

A total of 510 samples were included in the clinical study for FLC Kappa and FLC Lambda assays. This study included 177 samples from patients diagnosed with multiple myeloma and 144 samples from patients diagnosed with AL amyloidosis, and 189 non-myelomalnon-amyloidosis subjects with various clinical conditions.

The levels of Kappa free light chains and Lambda free light chains were measured in the serum samples using the FLC Kappa and the FLC Lambda kits for the calculation of the [Kappa FLC] / [Lambda FLC] ratio of each sample.

The clinical sensitivity calculated using [Kappa FLC] / [Lambda FLC] ratio for the clinical diagnosis of Multiple Myeloma (n=366) is as follows:

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	Clinical diagnosis of Multiple Myeloma
	Positive	Negative	Total
Sebia FLC Kappa and Lambda ratio	Positive	171	28	199
	Negative	6	161	167
	Total	177	189	366

Clinical sensitivity: 96.6% (95% confidence interval: 94.0% to 99.3%) Clinical specificity: 85.1% (95% confidence interval: 79.4% to 89.5%)

The Clinical sensitivity using [Kappa FLC] / [Lambda FLC] ratio for the clinical diagnosis of AL Amyloidosis (n=333) is as follows:

		Clinical diagnosis of AL Amyloidosis
		Positive	Negative	Total
Sebia FLC	Positive	131	28	159
Kappa and Lambda	Negative	13	161	174
ratio	Total	144	189	333

Clinical sensitivity: 91.0% (95% confidence interval: 86.3% to 95.7%) Clinical specificity: 85.1% (95% confidence interval: 79.4% to 89.5%)

7. CONCLUSION

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

Regulation Number and Section

§ 866.5550 Immunoglobulin (light chain specific) immunological test system.

(a)
Identification. An immunoglobulin (light chain specific) immunological test system is a device that consists of the reagents used to measure by immunochemical techniques both kappa and lambda types of light chain portions of immunoglobulin molecules in serum, other body fluids, and tissues. In some disease states, an excess of light chains are produced by the antibody-forming cells. These free light chains, unassociated with gamma globulin molecules, can be found in a patient's body fluids and tissues. Measurement of the various amounts of the different types of light chains aids in the diagnosis of multiple myeloma (cancer of antibody-forming cells), lymphocytic neoplasms (cancer of lymphoid tissue), Waldenstrom's macroglobulinemia (increased production of large immunoglobulins), and connective tissue diseases such as rheumatoid arthritis or systemic lupus erythematosus.(b)
Classification. Class II (performance standards).