HEVYLITE HUMAN IGM KAPPA AND LAMBDA KIT FOR USE ON SPAPLUS

K140686 · The Binding Site Group , Ltd. · PDE · Aug 6, 2014 · Immunology

Device Facts

• Record ID: K140686
• Device Name: HEVYLITE HUMAN IGM KAPPA AND LAMBDA KIT FOR USE ON SPAPLUS
• Applicant: The Binding Site Group , Ltd.
• Product Code: PDE · Immunology
• Decision Date: Aug 6, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5510
• Device Class: Class 2

Intended Use

Hevylite™ Human IgM Kappa Kit for use on SPAPLUS®: Hevylite Human IgM Kappa Kit for use on SPAPLUS is intended for the in vitro quantification of IgM Kappa (combined μ heavy and κ light chain) concentration in human serum on the SPAPLUS. The test result is to be used with previously diagnosed Waldenstrom’s macroglobulinemia in conjunction with other clinical and laboratory findings. This assay has not been established for the diagnosis, monitoring and prognosis of Waldenstrom’s macroglobulinemia. Hevylite™ Human IgM Lambda Kit for use on SPAPLUS®: Hevylite Human IgM Lambda Kit for use on SPAPLUS is intended for the in vitro quantification of IgM Lambda (combined μ heavy and λ light chain) concentration in human serum on the SPAPLUS. The test result is to be used with previously diagnosed Waldenstrom’s macroglobulinemia in conjunction with other clinical and laboratory findings. This assay has not been established for the diagnosis, monitoring and prognosis of Waldenstrom’s macroglobulinemia.

Device Story

Hevylite Human IgM Kappa and Lambda kits are in-vitro diagnostic assays for use on the Binding Site SPAPLUS analyzer. The device quantifies IgM kappa and IgM lambda concentrations in human serum using turbidimetric technology. Samples are mixed with sheep anti-IgM kappa or anti-IgM lambda antibodies coated onto polystyrene latex particles. As immune complexes form, light scatter increases; the analyzer measures the decrease in intensity of the incident light beam. The system uses an excess of antibody, ensuring immune complex formation is proportional to antigen concentration. Results are calculated against a calibration curve. The device is intended for use in clinical laboratories by trained personnel. Output provides quantitative concentration values used by clinicians in conjunction with other clinical findings for patients with previously diagnosed Waldenström's macroglobulinaemia. The device aids in assessing disease status by providing specific immunoglobulin light chain measurements.

Clinical Evidence

No clinical diagnostic studies performed. Analytical performance validated via bench testing: precision (total CV ≤ 10%), linearity (R² ≥ 0.999), and method comparison against predicate (N=227-269 samples). Agreement studies showed positive/negative agreement > 89% for IgMκ/λ and ratios. Reference ranges established using 147 healthy adult donors.

Technological Characteristics

Turbidimetric assay; sheep anti-IgM kappa/lambda antibodies coated on polystyrene latex particles. Energy source: light beam for scatter measurement. Platform: Binding Site SPAPLUS analyzer. Connectivity: automated analyzer. Calibration: traceable to ERM-DA470k. Software: automated instrument control and data processing.

Indications for Use

Indicated for in-vitro quantification of IgM kappa and IgM lambda concentrations in human serum for patients with previously diagnosed Waldenström's macroglobulinaemia. Not indicated for the diagnosis, monitoring, or prognosis of Waldenström's macroglobulinaemia.

Regulatory Classification

Identification

An immunoglobulins A, G, M, D, and E immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the immunoglobulins A, G, M, D, an E (serum antibodies) in serum. Measurement of these immunoglobulins aids in the diagnosis of abnormal protein metabolism and the body's lack of ability to resist infectious agents.

Predicate Devices

• Hevylite™ Human IgM Kappa Kit for use on Siemens BN™ II Systems (k113823)
• Hevylite™ Human IgM Lambda Kit for use on Siemens BN™ II Systems (k113823)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized graphic of an eagle or bird-like figure with three curved lines representing its body and wings. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circular fashion around the graphic.

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

August 6, 2014

THE BINDING SITE GROUP LTD C/O MS SUZANNE HORNE, REGULATORY AFFAIRS MANAGER 8 CALTHORPE ROAD, EDGBASTON BIRMINGHAM, WEST MIDLANDS B15 10T United Kingdom

Re: K140686

Trade/Device Name: Hevylite Human IgM Kappa Kit For Use On SPAPLIS® Hevylite Human IgM Lambda Kit For Use On SPAPLUS® Regulation Number: 21 CFR 866.5510 Regulation Name: Immunoglobulins A, G, M, D, and E immunological test system Regulatory Class: II Product Code: PDE, PDF Dated: July 04, 2014 Received: July 07, 2014

Dear Ms. Horne:

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. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. 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

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely vours.

# Maria M. Chan -S

Maria M. Chan, Ph.D. Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) K140686

#### Device Name

Hevylite Human IgM Kappa Kit for use on SPAPLUS Hevylite Human IgM Lambda Kit for use on SPAPLUS

#### Indications for Use (Describe)

Hevylite Human IgM Kappa kit for use on SPAPLUS is intended for the in-vitro quantification of IgM kappa (combined u heavy and k light chain) concentration in human serum on the Binding Site SPAPLUS. The test result is to be used with previously diagnosed Waldenström's macroglobulinaemia in conjunction with other clinical findings. This assay has not been established for the diagnosis of Waldenstrom's macroglobulinaemia.

Hevylite Human IgM Lambda kit for use on SPAPLUS is intended for the in-vitro quantification of IgM lambda (combined u heavy and A light chain) in human serum on the Binding Site SPAPLUS. The test result is to be used with previously diagnosed Waldenstrom's macroglobulinaemia. The test result is to be used in conjunction with other clinical findings.

This assay has not been established for the diagnosis, monitoring and prognosis of Waldenström's macroglobulinaemia.

#### Type of Use (Select one or both, as applicable)

2 Prescription Use (Part 21 CFR 801 Subpart D)

_ Over-The-Counter Use (21 CFR 801 Subpart C)

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#### FOR FDA USE ONLY

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# Hevylite® Human IgM Kappa and IgM Lambda Kits for use on SPAPLUS®

# 510(k) Summarv

Version: 5th August 2014

Contact Information: Suzanne Horne The Binding Site 8 Calthorpe Road Edgbaston Birmingham, B15 1QT West Midlands U.K Telephone: [phone] Email: [e-mail]

Hevylite® and SPAPLUS® are registered trademarks of the Binding Site Group Ltd, Birmingham, UK. BN™II is a trademark of Siemens Healthcare Diagnostics Inc.

#### Trade Names:

Hevylite® Human IgM Kappa Kit for use on SPAPLUS® Hevylite® Human IgM Lambda Kit for use on SPAPLUS®

#### Classification Names:

| Product<br>Classification<br>Code | Product Code Name | Device<br>Class | Classification<br>Panel | 21 CFR Section |
|-----------------------------------|--------------------------------------------------------------|-----------------|-------------------------|----------------|
| PDE | Immunoglobulin M Kappa<br>Heavy and light chain<br>combined | II | Immunology | 866.5510 |
| PDF | Immunoglobulin M Lambda<br>heavy and light chain<br>combined | II | Immunology | 866.5510 |

#### Intended use:

Hevylite Human IgM Kappa Kit for use on SPAPLUS is intended for the in vitro quantification of IgM Kappa (combined u heavy and k light chain) concentration in human serum on SPAPLUS. The test result is to be used with previously diagnosed Waldenström's macroglobulinaemia in conjunction with other clinical and laboratory findings.

This assay has not been established for the diagnosis, monitoring and prognosis of Waldenstrom's macroglobulinaemia.

Hevylite Human IgM Lambda Kit for use on SPAPLUS is intended for the in vitro quantification of IgM Lambda (combined u heavy and À light chain) concentration in human serum on SPAPLUS. The test

Image /page/3/Picture/13 description: The image contains the words "Binding Site" in bold, black font. To the right of the words is a logo that contains a yellow circle with a black silhouette of the British Isles. Below the circle are two black arms that are holding up the circle. Below the arms is a yellow rectangle with the words "The Binding Site" in black font.

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result is to be used with previously diagnosed Waldenstrom's macroglobulinaemia in conjunction with other clinical and laboratory findings.

This assay has not been established for the diagnosis, monitoring and prognosis of Waldenstrom's macroglobulinaemia.

#### Test Principle:

Evaluating the concentration of a soluble antigen (e.g. IgM Kappa and IgM Lambda) by turbidimetry involves the addition of the test sample to a solution containing the appropriate antibody (anti-IgM kappa and anti-IgM lambda) in a reaction vessel or cuvette. A beam of light id passed through the cuvette and, as the antigen-antibody reaction proceeds, the light passing through the cuvette is scattered increasingly as insoluble immune complexes are formed. Light scatter is monitored by measuring the decrease in intensity of the incident beam of light. The antibody in the cuvette is in excess so the amount of immune complex formed is proportional to the antigen concentration. A series of calibrators of known antigen concentration are assayed initially to produce a calibration curve of measured light scatter versus antigen concentration. Samples of unknown antigen concentration can then be assayed and the results read from the calibration curve.

#### Substantial Equivalence Information:

Predicate device: Hevylite Human IgM Kappa and IgM Lambda Kits for use on Siemens BN™II Systems (k113823)

#### Comparison with Predicate:

Similarities

| Item | Device | Predicate |
|---------------------|-------------------------------------------------------------------------------|-------------------------------------------------------------------------------|
| Intended use | <i>In-vitro</i> quantification of IgM kappa and IgM Lambda | <i>In-vitro</i> quantification of IgM kappa and IgM Lambda |
| Specimen Type | Serum | Serum |
| Antibody | Sheep anti-IgM kappa and anti-<br>IgM lambda coated onto<br>polystyrene latex | Sheep anti-IgM kappa and anti-<br>IgM lambda coated onto<br>polystyrene latex |
| Open Vial Stability | 1 month | 1 month |

#### Differences

| Item | Device | Predicate |
|-----------------------------|------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------|
| Standard Measuring<br>range | IgM Kappa: 0.2 -5.0g/L (1/10)<br>IgM Lambda: 0.18 – 4.50g/L<br>(1/10) | IgM Kappa: 0.2 – 6.4g/L (1/100)<br>IgM Lambda: 0.175-5.60g/L<br>(1/100) |
| Reference Interval | IgM Kappa: 0.19 – 1.63g/L<br>IgM Lambda: 0.12 – 1.01g/L<br>IgM Kappa/Lambda: 1.18 – 2.74 | IgM Kappa: 0.29 – 1.82g/L<br>IgM Lambda: 0.17 - 0.94g/L<br>IgM Kappa/Lambda: 0.96 – 2.30 |
| Method | Turbidimetry | Nephelometry |
| Instrument | Binding Site SPAPLUS | Siemens Behring Nephelometer II<br>(BNTMII) |

#### Discussion:

The differences between the methods of the devices do not affect the safety and effectiveness as they both measure light scatter, the only difference is where the detector is in the light source.

Image /page/4/Picture/14 description: The image contains the words "Binding Site" in large, bold, black font. To the right of the words is a logo that contains a yellow circle with a black silhouette of the United Kingdom inside. Below the circle are two black hands holding the circle up. Below the hands is a yellow rectangle with the words "The Binding Site" written in black font.

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The differences in the measuring ranges do not affect the safely and effectiveness as they both have the same lower limit and any results >5.0g/L on the kappa kit and >4.5g/L on the lambda kit will not have a clinical impact on patients if the results were quantified on the BN™II kit or reported as >5.0g/L on the kappa kit and >4.5g/L on the lambda kit.

The differences in the reference intervals do not affect the safety and effectiveness as the measuring ranges for the SPAPLUS kits were generated and the measuring ranges on the BN™II kits were from literature with different samples. The comparison data shows that the results obtained on both sets of kits are equivalent to each other.

#### Performance Characteristics:

#### Analytical performance:

# Precision/Reproducibility:

The precision study was based on CLSI (EP5-A2) Evaluation of Precision Performance of Quantitative Measurement Methods. Precision was initially evaluated using three samples of processed sera pooled with fully preserved citrate beta alanine that together had analyte levels that spanned measuring range of the assays (IgM Kappa 0.2 - 5.0g/L, IgM Lambda: 0.18 - 4.50g/L).

The 21 day precision study was performed by running the sera samples in duplicate (within-run analysis), two runs per day (between-run analysis) over 21 days (between-day) using three reagent lots (Batches 1, 2 and 3) and four instruments (SPAPLUS 2, 5, 7 and 9).

An additional "bridging" precision study was carried out. Precision was evaluated using five samples of pooled native sera that together had analyte levels that spanned the measuring range of the assays (IgM Kappa 0.2 - 5.0g/L, IgM Lambda: 0.18 - 4.50g/L). A summary of the precision samples used are described below:

The 21 day bridging precision study was performed by running the sera samples in duplicate (within-run analysis), two runs per day (between-run analysis) over 21 days (between-day) using one reagent lot and three instruments (SPAPLUS 1, 9 and 12).

# Repeatability

Initial IgM Kappa (Data present in IFU):

| Sample | N | Mean | Within Run | | Between run | | Between day | | Total | | Pass? (CV<8%) |
|------------|----|------|------------|-----|-------------|-----|-------------|-----|-------|-----|---------------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | |
| High Level | 84 | 4.13 | 0.08 | 1.8 | 0.08 | 1.8 | 0.21 | 4.6 | 0.24 | 5.3 | Pass |
| Mid Level | 84 | 1.80 | 0.03 | 1.5 | 0.03 | 1.3 | 0.66 | 3.5 | 0.08 | 4.1 | Pass |
| Low Level | 84 | 0.34 | 0.01 | 2.4 | 0.01 | 3.3 | 0.02 | 4.9 | 0.02 | 6.4 | Pass |

# IgM Bridging Study:

| Sample | N | Mean | Within Run | | Between run | | Between day | | Total | | Pass? |
|--------|----|-------|------------|-----|-------------|-----|-------------|-----|-------|-----|---------|
| | | (g/L) | SD | %CV | SD | %CV | SD | %CV | SD | %CV | (CV<8%) |
| 1 | 84 | 0.34 | 0.01 | 1.8 | 0.01 | 2.7 | 0.01 | 3.2 | 0.02 | 4.6 | Pass |
| 2 | 84 | 1.13 | 0.16 | 1.4 | 0.03 | 2.3 | 0.02 | 1.9 | 0.03 | 3.3 | Pass |
| 3 | 84 | 1.89 | 0.28 | 1.8 | 0.04 | 2.3 | 0.07 | 4.6 | 0.08 | 5.4 | Pass |
| 4 | 84 | 4.50 | 0.04 | 0.9 | 0.07 | 1.5 | 0.09 | 2.1 | 0.12 | 2.8 | Pass |
| 5 | 84 | 5.20 | 0.05 | 1.0 | 0.11 | 2.1 | 0.14 | 2.5 | 0.19 | 3.5 | Pass |

Image /page/5/Picture/15 description: The image contains the words "Binding Site" in bold, black font. To the right of the words is an image of two hands holding a yellow circle with a black silhouette of the United Kingdom. Below the hands is a yellow rectangle with the words "The Binding Site" in black font.

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#### lnitial IgM Lambda (Data present in IFU):

| Sample | N | Mean | Within Run | | Between run | | Between day | | Total | | Pass? (CV<8%) |
|------------|----|------|------------|-----|-------------|-----|-------------|-----|-------|-----|---------------|
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | |
| High Level | 84 | 4.11 | 0.07 | 1.7 | 0.06 | 1.5 | 0.18 | 4.4 | 0.20 | 5.0 | Pass |
| Mid Level | 84 | 0.96 | 0.02 | 1.9 | 0.01 | 0.6 | 0.03 | 3.2 | 0.03 | 3.8 | Pass |
| Low Level | 84 | 0.29 | 0.01 | 2.0 | 0.01 | 2.1 | 0.02 | 5.4 | 0.02 | 6.1 | Pass |

#### lgM Lambda Bridging Study:

| Sample | N | Mean (g/L) | Within Run SD | Within Run %CV | Between run SD | Between run %CV | Between day SD | Between day %CV | Total SD | Total %CV | Pass? (CV<8%) |
|--------|----|------------|---------------|----------------|----------------|-----------------|----------------|-----------------|----------|-----------|---------------|
| 1 | 84 | 0.26 | 0.01 | 2.0 | 0.01 | 3.3 | 0.01 | 4.1 | 0.01 | 5.7 | Pass |
| 2 | 84 | 0.71 | 0.01 | 1.3 | 0.02 | 3.0 | 0.00 | 0.0 | 0.02 | 3.3 | Pass |
| 3 | 84 | 1.22 | 0.01 | 1.0 | 0.07 | 5.8 | 0.00 | 0.0 | 0.07 | 5.9 | Pass |
| 4 | 84 | 3.86 | 0.05 | 1.3 | 0.16 | 4.2 | 0.10 | 2.6 | 0.20 | 5.1 | Pass |
| 5 | 84 | 5.24 | 0.06 | 1.1 | 0.34 | 6.5 | 0.19 | 3.6 | 0.39 | 7.5 | Pass |

# Reproducibility

#### Initial IgM Kappa Data:

| Sample | Mean<br>(g/L) | #<br>Runs | #<br>Days | Within-<br>Run | | Between-<br>run | | Between-<br>Instrument | | Between-<br>Batch | | Total | | Pass?<br>(CV<8%) |
|---------------|---------------|-----------|-----------|----------------|---------|-----------------|---------|------------------------|---------|-------------------|---------|-------|---------|------------------|
| | | | | SD | CV<br>% | SD | CV<br>% | SD | CV<br>% | SD | CV<br>% | SD | CV<br>% | |
| High<br>Level | 4.13 | 2 | 21 | 0.08 | 1.8 | 0.08 | 1.8 | 0.15 | 3.6 | 0.22 | 5.4 | 0.24 | 5.3 | Pass |
| Mid Level | 1.80 | 2 | 21 | 0.03 | 1.5 | 0.03 | 1.3 | 0.06 | 3.1 | 0.06 | 3.3 | 0.08 | 4.1 | Pass |
| Low<br>Level | 0.34 | 2 | 21 | 0.01 | 2.4 | 0.01 | 3.3 | 0.01 | 3.8 | 0.00 | 0.3 | 0.02 | 6.4 | Pass |

#### IgM Kappa Bridging Study:

| Sample | Mean (g/L) | # Runs | # Days | Within-Run | | Between-run | | Between-Instrument | | Total | | Pass?<br>(CV<8%) |
|--------|------------|--------|--------|------------|-----|-------------|-----|--------------------|-----|-------|-----|------------------|
| | | | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | |
| 1 | 0.34 | 2 | 21 | 0.01 | 1.8 | 0.01 | 2.7 | 0.01 | 2.7 | 0.02 | 4.6 | Pass |
| 2 | 1.13 | 2 | 21 | 0.16 | 1.4 | 0.03 | 2.3 | 0.02 | 1.9 | 0.03 | 3.3 | Pass |
| 3 | 1.89 | 2 | 21 | 0.28 | 1.8 | 0.04 | 2.3 | 0.07 | 3.6 | 0.08 | 5.4 | Pass |
| 4 | 4.50 | 2 | 21 | 0.04 | 0.9 | 0.07 | 1.5 | 0.07 | 1.6 | 0.12 | 2.8 | Pass |
| 5 | 5.20 | 2 | 21 | 0.05 | 1.0 | 0.11 | 2.1 | 0.07 | 1.3 | 0.19 | 3.5 | Pass |

#### Initial IgM Lambda Data:

| Sample | Mean<br>(g/L) | #<br>Runs | #<br>Days | Within-Run | | Between-run | | Between-Instrument | | Between-Batch | | Total | | Pass?<br>(CV<8%) |
|---------------|---------------|-----------|-----------|------------|------|-------------|------|--------------------|------|---------------|------|-------|-----|------------------|
| | | | | SD | CV % | SD | CV % | SD | CV % | SD | CV % | SD | CV% | |
| High<br>Level | 4.11 | 2 | 21 | 0.07 | 1.7 | 0.06 | 1.5 | 0.16 | 3.9 | 0.11 | 2.7 | 0.20 | 5.0 | Pass |
| Mid Level | 0.96 | 2 | 21 | 0.02 | 1.9 | 0.01 | 0.6 | 0.03 | 3.4 | 0.02 | 2.3 | 0.03 | 3.8 | Pass |
| Low<br>Level | 0.29 | 2 | 21 | 0.01 | 2.0 | 0.01 | 2.1 | 0.02 | 5.0 | 0.01 | 3.3 | 0.02 | 6.1 | Pass |

Image /page/6/Picture/12 description: The image shows the words "Binding Site" in a bold, sans-serif font. To the right of the words is a logo that features a yellow circle with a black silhouette of the British Isles inside. The circle is being held up by two black hands. Below the circle is a yellow rectangle with the words "The Binding Site" in black font.

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| Sample | Mean<br>(g/L) | #<br>Runs | #<br>Days | Within-Run | | Between-<br>run | | Between-<br>Instrument | | Total | | Pass?<br>(CV<8%) |
|--------|---------------|-----------|-----------|------------|-----|-----------------|-----|------------------------|-----|-------|-----|------------------|
| | | | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | |
| 1 | 0.26 | 2 | 21 | 0.01 | 2.0 | 0.01 | 3.3 | 0.01 | 4.7 | 0.01 | 5.7 | Pass |
| 2 | 0.71 | 2 | 21 | 0.01 | 1.3 | 0.02 | 3.0 | 0.01 | 0.8 | 0.02 | 3.3 | Pass |
| 3 | 1.22 | 2 | 21 | 0.01 | 1.0 | 0.07 | 5.8 | 0.02 | 2.0 | 0.07 | 5.9 | Pass |
| 4 | 3.86 | 2 | 21 | 0.05 | 1.3 | 0.16 | 4.2 | 0.14 | 3.5 | 0.20 | 5.1 | Pass |
| 5 | 5.24 | 2 | 21 | 0.06 | 1.1 | 0.34 | 6.5 | 0.33 | 6.3 | 0.39 | 7.5 | Pass |

# IgM Lambda Bridging Study:

# Linearity/assay reportable range:

The linearity of the assays was assessed using one lot of reagent on one analyser.

The high pools for IgM kappa and IgM Lambda were prepared from a serum sample with a naturally high concentration of IgM kappa and adjusted by the addition of purified IgM. A low pool for IgM kappa was prepared from a normal serum sample adjusted with the addition of IgM depleted serum. The low pool for IgM Lambda was prepared from IgM depleted serum. A dilution series was prepared for IgM kappa and IgM lambda separately by blending the respective high and low pool (described above); to produce a total of 12 concentrations (described below) that covered the measuring range of the assays. Three replicates of each level of the dilution series were run and the mean calculated.

An additional study was performed to evaluate the bottom of the standard measuring range for IgM Kappa. A high pool was prepared from pooled normal human serum and diluted with IgM depleted serum to provide samples with a concentration range of 0.086 - 0.405g/L.

Linearity was evaluated by calculating the percentage recovery at each concentration in the dilution series, and the %CV of the 3 replicates. The acceptance criteria was a mean recovery of ≤±10%, or an acceptable recovery of ≤±16.8% and a CV of <8%. Linearity was demonstrated at the concentrations spanning the claimed measuring range. The observed values were graphed against the calculated values and a linear regression was performed.

The regression plot equations where y is the measured level of IgM kappa or lambda and x is the theoretical concentration were:

| Sample | Dilution Range<br>(g/L) | Regression<br>equation | Slope<br>(95% CI) | Y-Intercept (95%<br>CI) | R |
|----------------|-------------------------|------------------------|------------------------|-------------------------|-------|
| IgMκ<br>Sample | 0.158 – 5.911 | y = 1.00x + 0.02 | 1.00<br>(0.97 to 1.03) | 0.02<br>(-0.08 to 0.12) | 0.999 |
| IgMκ<br>Sample | 0.086 – 0.405 | y = 1.02 – 0.00 | 1.02<br>(0.95 to 1.08) | 0.00<br>(-0.02 to 0.01) | 0.997 |
| IgMλ<br>Sample | 0.131 – 5.085 | y = 0.97x + 0.02 | 0.97<br>(0.96 to 0.99) | 0.02<br>(-0.03 to 0.07) | 1.00 |

The approximate measuring range of the Hevylite Human IgM Kappa kit for use on the SPAPLUS is 0.2 -5.0g/L.

The approximate measuring range of the Hevylite IgM Lambda kit for use on the SPAPLUS is 0.18 -4.50g/L.

Image /page/7/Picture/12 description: The image contains the words "Binding Site" in large, bold, black font. To the right of the words is a logo that features two hands holding up a yellow circle. Inside the circle are two animals, possibly a dog and a cat. Below the hands and circle is a yellow rectangle with the words "The Binding Site" written in black font.

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#### Linecision:

The precision and accuracy of the SPAPLUS analysers' instrument dilution function, a "linecision" study was carried out on one analyser and one reagent lot.

Two serum samples with target analyte concentrations falling in the overlapping regions between the measuring ranges of each instrument dilution. Each sample was run with 20 aspirations against each instrument measuring range (1/1, 1/10, 1/90).

An additional study was carried out to evaluate the manual pre-dilution step using 3 separate users. A third sample was targeted at the overlapping region between the 1/90 and 1/250 measuring ranges. The third sample was run with 20 aspirations against the 1/90 instrument dilution. When sample three was run at the 1/250 instrument dilution it was carried out by 3 operators who diluted the sample as per the product insert. That sample was then aspirated 20 times against the 1/250 instrument dilution.

The acceptance criteria were a recovery of ±10% for each sample and a CV of the 20 aspirations of ≤8%.

Traceability, Stability, Expected values (controls, calibrators, or methods):

#### Traceability

The calibrators, the master calibrator and controls are traceable to ERM-DA470k International Reference Material. The master calibrator is prepared from pooled human sera and is used to control calibration between lots.

#### Device Stability

Real-time stability studies were performed to support the following stability claims of the Hevylite IgM Kappa and Hevylite IgM Lambda kits:

- Shelf-life stability of the opened kits when stored at 2 - 8°C is up to 2 months.
- . Shelf-life stability for unopened kits when stored at 2 - 8°C is 12 months.

#### Detection limit:

The limit of blank (LoB) and limit of detection studies were performed based on the protocol described in CLSI EP17-A. The detection limit was determined by testing a blank sample (IgM depleted serum), samples close to the bottom of the neat 1/1 measuring range (LoQ), and a sample with value close to the blank (LoD) sample at a neat sample dilution (1/1).

The LoB was determined non-parametrically by running the IgM depleted sample. The assigned concentrations were equivalent to 0.00g/L for both IgM Kappa and IgM Lambda as the sample was depleted of analyte. Samples were tested 60 times to determine LoB.

The LoQ samples were five serum samples that had a mean analyte concentration within ±10% of the bottom of the measuring range. The assigned concentrations were equivalent to the neat measuring range (1/1), 0.02g/L for IgM kappa and 0.018g/L for IgM lambda. The five samples were tested 12 times each over 5 days for LoQ.

The LoD is the lowest measurable concentration of the analyte that can be distinguished from zero; it has been calculated as 0.0012g/L for IgM kappa and 0.0016g/L for IgM lambda for the minimum sample

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dilution for the assay (1/1). LoD was determined from the LoB value (LoD = LoB + 1.645 SDg where, SDJ is the pooled precision estimate of the LoQ study samples).

#### Analytical specificity:

#### Interfering Substances

Interference by endogenous substances were evaluated by addition of haemoglobin (5g/L), bilirubin (200mg/L), Intralipid (500mg/dL) and triglycerides (1000mg/dL) to test serum samples representing the analyte concentrations representing concentrations in the normal range, sample close to the lower medical decision point (LMDP), close to the upper medical decision point (UMDP), a low pathological sample and a high pathological sample. The negative control pools were prepared by spiking commercially obtained blank or saline when blank samples were not obtainable, into the sera base pools. Samples were tested three times each. The acceptance criteria was percentage difference of < ±10%. No significant interference was observed with the interferents tested.

Rheumatoid factor (RF) was not evaluated because this autoantibody is directed against the Fc portion of immunoglobulins. Interference is unlikely to take place in the Hevylite Human IgM kappa and IgM Lambda assays, as the Fc region of the anti-IgM kappa and antibodies are cleaved prior to coating onto the latex bead.

The medical decision points are defined as the upper and lower limits of the normal reference range.

The package insert states in the Limitations section that "Turbidimetric assays are not suitable for measurement of highly lipaemic or haemolysed samples containing high levels of circulating immune complexes (CICs) due to the unpredictable degree of non-specific scatter these sample types may generate. Unexpected results should be confirmed using an alternative assay method".

#### Cross reactivity:

Cross reactivity studies were carried out by testing Hevylite IgM Kappa and Hevylite IgM Lambda assays in the presence of high concentrations of potentially cross reacting monoclonal proteins in samples from IgA1K, IgA2K, IgA2A, IgG1K, IgG1A, IgG1A, IgG2K, IgG3K, IgG3A, IgG4A, IgG4A, K free light chain and λ free light chain multiple myeloma patient sera. No significant cross reactivity was observed.

The samples were all tested for total IgA, total IgA, total IgM, and also with the Hevylite IgM Kappa and IgM Lambda assays. The results for total IgM were compared with the results obtained by the Hevylite lgM Kappa and IgM Lambda assays.

In addition, IgMx patient samples were tested on Hevylite IgM Lambda kits to investigate potential crossreactivity, and similarly, IgMA patient samples were tested on IgM kappa kits.

#### Antigen excess effect:

Prozone parameters are in effect to protect the SPAPLUS analyser from antigen excess effects. Reaction kinetics of high level samples was compared to that of the top calibrator for each kit. Samples detected as being in excess are flagged with a "P" flag.

#### Assay cut-off:

The cut-off values are the reference ranges for the normal population which have been established from the reference range study.

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#### Comparison study:

#### Method comparison with predicate device:

A total of 227 and 269 sera samples spanning the range were assayed in singlicate on both Hevylite Human IgM kappa and IgM Lambda Kits for use on SPAPLUS respectively, these results were compared to those obtained on the Hevylite Human IgM Kappa and IgM Lambda Kits for use on Siemens BNII.

The serum samples included 41 Waldenström's Macroglobuliaemia patients, 23 normal donors and samples with elevated IgM Kappa and IgM Lambda levels. Passing and Bablok regression are based on the balance of the paired results:

#### lgM Kappa: y=0.84x + 0.07g/L

# lgM Lambda: y= 0.93x + 0.00g/L

The percentage positive (abnormal result) and negative (normal result) agreement in the serum samples for each assay:

| IgM Kappa | Predicate | |
|-----------|-----------|----------|
| | Positive | Negative |
| Positive | 159 | 2 |
| Negative | 11 | 55 |

Positive percentage agreement: 93.5% (88.7 to 96.7%) Neqative percentage agreement: 96.5% (87.9 to 99.5%) Overall agreement: 94.3% (88.3 to 95.6%)

| IgM Lambda | Predicate | |
|------------|-----------|----------|
| | Positive | Negative |
| Positive | 131 | 2 |
| Negative | 16 | 120 |

Positive percentage agreement: 89.1% (82.9 to 93.7%) Negative percentage agreement: 98.4% (94.2 to 99.8%) Overall agreement: 93.3% (89.6 to 96.0%)

When considering the upper and the lower limits of the reference range as medical decision points for the IgM Kappa / IgM Lambda Ratio, the positive and negative agreement are as follows:

| IgM Kappa/ IgM Lambda | Predicate | |
|-----------------------|-----------|----------|
| | Positive | Negative |
| Positive | 164 | 4 |
| Negative | 14 | 40 |

Positive percentage agreement: 92.1% (87.2 to 95.6%) Negative percentage agreement: 90.9% (78.3 to 87.5%) Overall agreement: 91.9% (87.5 to 95.1%)

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#### Clinical Studies:

Clinical Sensitivity and Specificity:

Clinical cut-off:

Not applicable.

#### Expected values/ Reference range:

The normal ranges established in accordance with CLSI C28-A3 using 147 UK adult blood donors. The assays were performed on the SPAPLUS analyser. A non-parametric analysis of specimens for the distribution of IgMк and IgMÀ was performed.

#### Normal range results as included in IFU:

| Normal adult serum | Mean | Median | 95 Percentile Range |
|-----------------------------|------|--------|---------------------|
| IgM kappa (g/L) | 0.71 | 0.63 | 0.19 - 1.63 |
| IgM lambda (g/L) | 0.39 | 0.35 | 0.12 - 1.01 |
| IgM kappa/ IgM lambda ratio | 1.85 | 1.81 | 1.18 - 2.74 |

The upper and lower limits of the reference range for IgMk (0.19 – 1.63g/L), IgMA (0.12 – 1.01g/L) and the IgMk/λ ratio (1.18 – 2.74) are defined as the "cut-offs". Samples with a Hevylite result above any of these reference ranges, or below the lower cut-off are classified as abnormal.

The cut-offs have been validated by comparing percentage agreement between the predicate device and the Hevylite IgM kappa and IgM Lambda kits.

# Conclusion:

The Hevylite Human IgM Kappa and the Hevylite Human IgM Lambda kits for use on the SPAPLUS are substantially equivalent to the Hevylite Human IgM Kappa and IgM Lambda Kits for use on Siemens BN™II as they have the same intended use, technological characteristics, features and principles of operation as the Hevylite Human IgM Kappa and IgM Lambda Kits for use on Siemens BN™II that do not raise new questions of safety and effectiveness.

The validation data demonstrates that the performance characteristics also do not raise new questions of safety and effectiveness of the Hevylite Human IgM Kappa and IgM Lambda kits for use on the SPAPLUS.

The comparison and the validation of cut-off studies demonstrate that the results obtained on the Hevylite Human IgM Kappa and IgM Lambda kits for use on SPAPLUS are equivalent and the normal/abnormal outcomes determined by the reference intervals provide no adverse effects to the patient results.

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