SPAPLUS

K100179 · The Binding Site Group , Ltd. · CZW · Aug 13, 2010 · Immunology

Device Facts

• Record ID: K100179
• Device Name: SPAPLUS
• Applicant: The Binding Site Group , Ltd.
• Product Code: CZW · Immunology
• Decision Date: Aug 13, 2010
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5240
• Device Class: Class 2

Indications for Use

This kit is intended for the quantitative in vitro determination of human C3c in serum using the Binding Site SPAPLUS™ turbidimetric analyser. This test should be used in conjunction with other laboratory and clinical findings.

Device Story

The Human C3c Kit is an in vitro diagnostic reagent system designed for use on the Binding Site SPAPLUS™ turbidimetric analyzer. The device measures C3c concentrations in human serum samples. Operation involves the turbidimetric analysis of antigen-antibody reactions; the analyzer processes the sample and provides a quantitative result. The device is intended for use by laboratory professionals in clinical settings to assist in the assessment of complement components. Results are interpreted by clinicians alongside other laboratory and clinical findings to support patient diagnosis and management.

Clinical Evidence

Bench testing only. Precision studies (CLSI EP5-A2) showed total CVs < 7%. Linearity (CLSI EP6-A) confirmed performance across 0.03-3 g/L. Method comparison with the predicate using 92 retrospective human serum samples yielded a Passing and Bablok fit of y = 0.99x + 0.00 (R²=0.99). Interference testing showed no significant impact from bilirubin, hemoglobin, or chyle.

Technological Characteristics

Turbidimetric immunoassay for quantitative determination of C3c. Designed for use on the SPAPLUS™ analyzer. Class II device under 21 CFR 866.5240.

Indications for Use

Indicated for the quantitative in vitro determination of human C3c in serum to aid in the assessment of complement components in clinical laboratory settings.

Regulatory Classification

Identification

A complement components immunological test system is a device that consists of the reagents used to measure by immunochemical techniques complement components C1q , C1r , C1s , C2 , C3 , C4 , C5 , C6 , C7 , C8 , and C9 , in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these proteins aids in the diagnosis of immunologic disorders, especially those associated with deficiencies of complement components.

Submission Summary (Full Text)

{0}

1

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k100179

B. Purpose for Submission:
New Device

C. Measurand:
Human complement C3c

D. Type of Test:
Quantitative, turbidimetric.

E. Applicant:
The Binding Site, Ltd

F. Proprietary and Established Names:
Human C3c Kit for use on $\mathrm{SPAPLUS}^{\mathrm{TM}}$

G. Regulatory Information:

1. Regulation section:
21 CFR § 866.5240 Complement components immunological test system

2. Classification:
Class-II

3. Product code:
CZW, Complement C3, antigen, antiserum, control

4. Panel:
Immunology (82)

H. Intended Use:

1. Intended use(s):
The Human C3c Kit is intended for the quantitative in vitro determination of human C3c in serum using the Binding Site $\mathrm{SPAPLUS}^{\mathrm{TM}}$ turbidimetric analyser. The test should be used in conjunction with other laboratory and clinical findings.

2. Indication(s) for use:
Same as Intended use.

3. Special conditions for use statement(s):
For prescription use only.

4. Special instrument requirements:
$\mathrm{SPAPLUS}^{\mathrm{TM}}$ Turbidimetric Analyzer (manufactured as Clinical Chemistry Analyzer under the names Prestige 24i, Sirrus, MGC240 by Tokyo Boeki, Japan, cleared under k040958). The $\mathrm{SPAPLUS}$ is identical to the Prestige 24 clinical chemistry analyzer without the ISE module.

The software used by the $\mathrm{SPAPLUS}^{\mathrm{TM}}$ is the same as that used by the Sirrus and Prestige 24i instruments. A minor modification has been made to the standard sample dilution for this assay. The standard ample dilution has been defaulted to $1/10$ (rather than neat) for patient samples and control samples, with all auto rerun facility activated at $1/20$ if the results obtained is over-range. The final result and test dilution appears on the instrument panel and printout. No changes have been made to the instruments functionality.

{1}

Following a review of the Guidance for Content of Premarket Submission for Software Contained in Medical Devices the risk is assessed as 'minor level of concern'. These modifications were previously covered in k062372 'Freelite for use on the SPAPLUS'. No additional modifications have been made to the analyzer or software in order to run the C3c assay.

# I. Device Description:

Human C3c Kit for Use on the $\mathrm{SPAPLUS}^{\mathrm{TM}}$ test includes:

1. Sheep anti-human C3c serum.
2. Calibrators and controls (from control pooled human serum samples referenced to DA470k international reference material).
3. Sample diluent.

# J. Substantial Equivalence Information:

1. Predicate device name(s):

Roche Diagnostics Corp, Tina Quant Complement C3c Test System.

2. Predicate 510(k) number(s):

k012361

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| | Device | Predicate |
| Item | Binding Site C3c SPAPLUS Kit | k012361 Tina-Quant C3c |
| Intended Use | For the quantitative in vitro determination of human C3c | Same |
| Method | Turbidometric immunoassay | Same |
| Pediatric use | No pediatric range | Same |
| Traceability | DA-470k | Same |
| Control | Normal and High level liquid ready-to-use controls | Same |
| Differences | | |
| --- | --- | --- |
| | Device | Predicate |
| Item | Binding Site C3c SPAPLUS Kit | k012361 Tina-Quant C3c |
| Sample type | Human Serum | Human Serum and Plasma |
| Instruments | SPAPLUS analyser | COBAS Integra analyzer |
| Measuring range | 0.25-3.0 g/L at 1/10 dilution | 0.11-6.0 g/L |
| Healthy Adult Reference Interval | Adults 0.81-1.57g/L | Adults 0.9-1.8g/L |
| LOD and LOQ | LOD = 0.012 g/L LOQ = 0.24 g/L | LOD = 0.11 g/L |
| Healthy Adult Reference Interval | Adults 0.81-1.57 g/L | Adults 0.9-1.8 g/L |
| Antibodies | Sheep (latexed) | Goat |
| Dose and dose of the drug | 1000 mg/kg | 1000 mg/kg |
| Dose and dose of the drug | 1000 mg/kg | 1000 mg/kg |

{2}

3

| Differences | | |
| --- | --- | --- |
| | Device | Predicate |
| Item | Binding Site C3c SPAPLUS Kit | k012361 Tina-Quant C3c |
| Calibrator and Controls | Provided in kit | Sold separately |
| Reagent Stability | Unopened: 3 months at 2-8°C
Opened (on-board) 28 days. | Opened (on-board) 42 days |

## K. Standard/Guidance Document Referenced (if applicable):

CLSI EP5-A2: Evaluation of Precision Performance of Quantitation Measurement Methods; Approved Guideline-Second Addition

CLSI EP6-A: Evaluation of the Linearity of Quantitative measurement procedures; A statistical approach; Approved Guideline

CLSI EPI7-A: Protocols for Determination of Limits of Detection and Limits of Quantitation, Approved Guideline.

Protocols for Determination of Limits of Detection and limits of Quantitation; Approved Guideline

## L. Test Principle:

Human C3c Kit for Use on the $\mathrm{SPAPLUS}^{\mathrm{TM}}$ test is a single-step immunoturbidimetric test. The assay determines concentration of C3c through photometric measurement of suspension formed (insoluble antigen-antibody complex agglutination) between antibodies to human C3c and C3c present in the sample. Concentrations are automatically calculated by reference to a calibration curve stored with in the instrument.

## M. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

#### a. Precision/Reproducibility:

The precision studies were performed following CLSI Evaluation of Precision Performance of Clinical Quantitative Measurement Methods; Approved Guideline (CLSI Document EP5-A2).

The study was carried out over 21 working days, with two runs per day. One user assessed three different samples (High, Medium and Low) using three different reagent lots on three different analyzers.

Results are summarized in the table below:

| Level | Mean
(g/L) | Within run | | Between run | | Between day | | Total precision | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| High | 3.114 g/L | 0.053 | 1.7% | 0.065 | 2.1% | 0.122 | 3.9% | 0.148 | 4.7% |
| Medium | 0.899 g/L | 0.017 | 2.0% | 0.023 | 2.6% | 0.028 | 3.2% | 0.039 | 4.6% |
| Low | 0.421 g/L | 0.009 | 2.5% | 0.015 | 4.0% | 0.017 | 4.7% | 0.025 | 6.6% |

All within-run, between-run and between day precision coefficients of variation are below 7%. The Total Precision for Sample 3 has the highest coefficient of variation at 6.6%. The Total Precision %CV for the medium level (Sample 2) is 4.6% and

{3}

4.7% for Sample 1 at the top end of the calibration curve. The study shows overall precision for the C3c SPAPLUS kit to be good.

## b. Linearity/assay reportable range:

Linearity was evaluated as per CLSI EP6-A (Evaluation of the Linearity of Quantitative measurement procedures; A statistical approach; Approved Guideline). Seven undiluted human serum samples (see table) previously identified as containing high levels of C3c and stored at -20°C were pooled to form a high linearity fluid.

This high level fluid was diluted with sample diluent (not reaction buffer) as it was not possible to obtain sufficient samples with adequate (low) levels to produce a low level linearity pool. Dilutions were prepared to cover the kit measuring range and each dilution was measured three times on three different kit lots at neat and a 1/10 sample dilution.

| Control | Assigned Conc. (g/L) | Results (g/L) | % Difference (+/-15% acceptance) |
| --- | --- | --- | --- |
| C3c Kit Lot-1: | | | |
| C3c High Control | 2.720 | 2.710 | -0.5 |
| C3c Low Control | 0.840 | 0.850 | 1.0 |
| | | 0.830 | -1.0 |
| Internal reference | 0.941 | 0.953 | 1.3 |
| | | 0.954 | 1.4 |
| C3 Kit Lot-2: | | | |
| C3c High Control | 2.730 | 2.660 | -2.6 |
| | | 2.660 | -1.3 |
| C3c Low Control | 0.790 | 0.770 | -2.4 |
| | | 0.750 | -4.8 |
| Internal reference | 0.941 | 0.901 | -4.3 |
| | | 0.918 | -4.8 |
| C3 Kit Lot-3: | | | |
| C3c High Control | 2.850 | 2.870 | 0.8 |
| | | 2.910 | 2.0 |
| C3c Low Control | 0.870 | 0.910 | 5.4 |
| | | 0.920 | 5.1 |
| Internal reference | 0.941 | 0.977 | 3.8 |
| | | 1.006 | 6.9 |

The results were plotted for each lot to inspect the linear relationship (batch-1 shown in figure below). In addition percentage recovery was calculated for the mean of each dilution.

{4}

5


| Linear - Term | Coefficient | SE | p |
| --- | --- | --- | --- |
| Intercept | -0.0080 | 0.0053 | 0.1349 |
| X | 0.9994 | 0.0037 | <0.0001 |
| Source of variation | SSq | DF | MSq | F | p |
| --- | --- | --- | --- | --- | --- |
| Linear Fit | 50.3503 | 1 | 50.3503 | 72081.27 | <0.0001 |
| Residual | 0.0342 | 49 | 0.0007 | - | - |
| Lack of Fit | 0.0115 | 15 | 0.0008 | 1.15 | 0.3555 |
| Pure error | 0.0227 | 34 | 0.0007 | | |
| Total | 50.3845 | 50 | | | |

This data confirms the linearity of the assay over the range of 0.03-3 g/L across 3 different kit lots.

Measuring range (reportable range) for C3s is 0.03 - 3 g/L for initial 1:10 dilution of samples that are automatically processed by the instrument. Samples with results in excess of 3 g/L are repeated on a higher dilution (1:20).

## Auto-dilution vs. manual dilution

Studies were done to support the accuracy and the precision of the SPAPLUS™ turbidimetric analyser automated sample dilution protocol, by comparing auto-dilution vs. manual-dilution. Three samples of different levels were tested on the SPAPLUS using off-line (manual) and on-line (auto) dilution method. The samples were all diluted in SPAPLUS instrument system diluent (SN080.S) which is the recommended diluent for this analyzer. Each sample dilution was tested 20 times and the mean, standard deviation and % coefficient of variation were calculated for each data set. In all three cases the manual (off-line) 1/20 dilution was compared to the 1/20 on-line dilution.

The test results are summarized in the table below:

{5}

| sample ID | 77 | | 97 | | 85 | |
| --- | --- | --- | --- | --- | --- | --- |
| SPA sample dilution | 1/1 | 1/20 | 1/1 | 1/20 | 1/1 | 1/20 |
| offline dilution | 1/20 | none | 1/20 | none | 1/20 | none |
| 1 | 3.160 | 3.208 | 2.400 | 2.351 | 1.260 | 1.301 |
| 2 | 3.180 | 3.178 | 2.420 | 2.414 | 1.300 | 1.320 |
| 3 | 3.060 | 3.137 | 2.360 | 2.381 | 1.240 | 1.288 |
| 4 | 3.160 | 3.197 | 2.380 | 2.412 | 1.320 | 1.317 |
| 5 | 3.100 | 3.149 | 2.320 | 2.417 | 1.260 | 1.294 |
| 6 | 3.160 | 3.228 | 2.420 | 2.393 | 1.320 | 1.315 |
| 7 | 3.100 | 3.159 | 2.400 | 2.800 | 1.280 | 1.307 |
| 8 | 3.200 | 3.128 | 2.380 | 2.371 | 1.320 | 1.267 |
| 9 | 3.060 | 3.088 | 2.400 | 2.329 | 1.260 | 1.316 |
| 10 | 3.200 | 3.069 | 2.440 | 2.369 | 1.320 | 1.279 |
| 11 | 3.120 | 3.121 | 2.360 | 2.395 | 1.260 | 1.313 |
| 12 | 3.160 | 3.190 | 2.440 | 2.353 | 1.300 | 1.302 |
| 13 | 3.120 | 3.188 | 2.380 | 2.400 | 1.260 | 1.200 |
| 14 | 3.160 | 3.188 | 2.440 | 2.348 | 1.300 | 1.280 |
| 15 | 3.100 | 3.231 | 2.380 | 2.395 | 1.240 | 1.300 |
| 16 | 3.140 | 3.184 | 2.420 | 2.399 | 1.300 | 1.280 |
| 17 | 3.100 | 3.199 | 2.400 | 2.417 | 1.220 | 1.274 |
| 18 | 3.160 | 3.140 | 2.460 | 2.343 | 1.280 | 1.254 |
| 19 | 3.160 | 3.151 | 2.420 | 2.387 | 1.240 | 1.282 |
| 20 | 3.100 | 3.207 | 2.420 | 2.435 | 1.280 | 1.281 |
| Mean | 3.135 | 3.167 | 2.402 | 2.405 | 1.278 | 1.289 |
| SD | 0.041 | 0.044 | 0.034 | 0.097 | 0.031 | 0.028 |
| CV | 1.32% | 1.39% | 1.40% | 4.04% | 2.43% | 2.16% |
| % diff from offline 1/20 | | 1.02% | | 0.14% | | 0.82% |

The within run precision show coefficient of variation below $5\%$ and the difference between the mean off-line and mean on-line dilution are $1\%$ or less. This study shows good agreement between the manual 1/20 and auto 1/20 sample dilutions.

# c. Traceability, Stability, Expected values (controls, calibrators, or methods): Controls and Calibrators

The calibrators and controls are manufactured from 'pooled human serum'. The control serum samples were traceable to European Reference Material DA 470. The kit C3c calibrators are assigned to an internal reference (IR) which is directly calibrated to the external reference standard DA-470. For the IR assignment both the IR fluid and DA-470 are tested at different dilutions across the curve (50%, 25%, 12.5% and 6.25%). All dilutions are tested in triplicate using 2 or more different kit batches on the SPAPLUS. A second set of dilutions are made and tested in the same way on a different SPAPLUS analyzer. From this information the IR value is calculated. The IR assignment is verified by testing it against DA-470 in triplicate using all assays that measure C3c.

{6}

The assayed controls have assigned values with a $+/-10\%$ in-house tolerance and target values for the low control and for the high control. The controls are assigned on calibration curves validated with the internal reference standard which is directly assigned to the international reference standard DA-470. The controls supplied with each lot are assayed on the kit and information on the values obtained, including the $+/-15\%$ customer range.

The comparison study provided show good agreement with no bias seen across the measuring range and a mean percentage difference between the two assays of $-1.1\%$

# Stability

Three kit lots of C3c, with each reagent originating from different batches, were tested after storage at $22^{\circ}\mathrm{C}$ for 1 week to simulate shipping conditions. The kits were tested at Time 0, 3, 7 and 10 months after storage at the recommended temperature of $2 - 8^{\circ}\mathrm{C}$ . At each stage a calibration curve was run together with the two kit controls and an internal reference (IR) standard was also run at time 0.

| Control | Assigned Conc. (g/L) | % (+8.5%) | % (+15%) | % (+15%) | % (+15%) |
| --- | --- | --- | --- | --- | --- |
| Lot-1: | | 0 months | 3 months | 7 months | 10 months |
| IR | 0.941 | 3.9 | | 0.7 | 5.2 |
| | | 2.9 | | 2.6 | 4.4 |
| High | 2.72 | -3.9 | -7.0 | -2.2 | 1.7 |
| | | -5.2 | -10.5 | -1.0 | 3.5 |
| Low | 0.84 | -2.0 | -7.9 | 3.8 | 10.2 |
| | | -2.9 | -10.7 | 2.0 | 11.5 |
| Lot-2: | | 0 months | 3 months | 7 months | 10 months |
| IR | 0.941 | -3.3 | | 0.1 | 3.1 |
| | | -3.3 | | -3.7 | 1.7 |
| High | 2.73 | -4.7 | -3.8 | 2.4 | 1.8 |
| | | -5.9 | -1.4 | 2.5 | 4.9 |
| Low | 0.79 | -3.9 | -6.6 | 7.1 | -2.9 |
| | | -2.4 | -6.2 | 9.2 | 0.3 |
| Lot-3: | | 0 months | 3 months | 7 months | 10 months |
| IR | 0.941 | -2.7 | | -4.7 | 1.1 |
| | | -4.3 | | -3.5 | 2.0 |
| High | 2.85 | -3.9 | -9.9 | -0.8 | 0.1 |
| | | -3.9 | -9.6 | -2.4 | 0.2 |
| Low | 0.87 | -6.3 | -0.6 | 2.8 | 0.8 |
| | | -3.3 | -0.5 | -3.3 | 2.9 |

The results obtained from Low and High Controls were within $\pm 15\%$ of the assigned value ( $\pm 8.5\%$ at time 0), showing stability of the Kit reagents for at least 3

{7}

8

months from the date of manufacture when stored at the recommended storage temperature of 2-8°C.

On-Board stability tests were performed by storing vials 'on-board' the SPAPLUS for a period of at least 28 days. Open vial Stability were tested by storing reagents in open vials for 3 months at 2 - 8°C. The results obtained from Low and High Controls were within ±15% of the assigned value.

d. Detection limit:

CLSI EPI7-A (Protocols for Determination of Limits of Detection and Limits of Quantitation, Approved Guideline) were employed for determining limit of detection. A blank, the bottom calibrator and a sample known to contain C3c, diluted to give a value just greater than the blank, were tested 60 times on the SPAPLUS analyzer. The standard deviation (SD) and the percentage coefficient of variation (%CV) were calculated in each case. The 60 blank absorbance readings gave a mean value of 0.0027 absorbance units with an SD of 0.0008 units.

The limit of quantitation (LoQ): The bottom calibrator point was the lowest point of the measuring range and therefore was considered as the limit of quantitation (LoQ). The LoQ was assigned a value of 0.24g/L for kit lot N0054.

The limit of blank (LoB): Below LoQ point, samples were flagged as below the measurable range, and no concentration value was calculated. LoB was estimated as the mean of blank +2 SD using the blank (sample diluent) as a zero calibrator. The results derived from 0.0027 + (0.0008 × 2) = 0.0040 abs units gave an estimated value of 0.005g/L (LoB).

The limit of detection (LoD): The limit of detection (LoD) was estimated as the mean value obtained for the very low level sample using the blank as a zero calibrator. The very low level sample (diluted sample) gave a mean value of 0.0088 absorbance units giving an estimated value of 0.012g/L which was considered to be the limit of detection (LoD).

e. Analytical specificity:

Cross-reactivity with other complement components:

The IEP and ouchterlony tests were performed to demonstrate the absence of cross reaction with other complement components including major split products (C4a etc.) and other subunits (C Iq etc.). Western blots analysis was used to demonstrate the absence of cross-reactivity to C3a and C3dg. Results showed the sheep anti-human C3c serum specifically binds to human C3c.

Interference – endogenous substances

Susceptibility to interference was assessed by adding a high concentration of a potential interferent to a test sample containing known concentrations of C3c. The method used to check for chyle, a mixture of biological lymph and chylomicrons (lipids and proteins), and haemoglobin was based on the Interference Check A plus™ (Sysmex, Japan). In the same way bilirubin interference was assessed by adding a high concentration of bilirubin to a test serum sample and comparing it to a blank. Percentage interference was calculated from comparison with the sample blank. Deviations less than or equal to +/-10% of the blank value were considered to show 'no significant interference'.

{8}

9

| | Bilirubin (200mg/mL) | Haemoglobin (5g/L) | Chyle (1500FTU) |
| --- | --- | --- | --- |
| Mean C3c (g/L) | 0.313 | 0.294 | 0.293 |
| % interference | 4.6% | 0.1% | 1% |

No significant interference was seen when high concentrations of haemoglobin (5g/L), bilirubin (200mg/L) and chyle (1500 FTU) were added to serum samples.

f. Assay cut-off:
Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

Method comparison between Binding Site's C3c SPAPLUS™ assays to the already 510k approved Tina-Quant C3c Test system (k012361 Roche Diagnostics Corp.) was done by using 92 human serum retrospective samples. The clinical samples were collected and stored at -80°C in order to preserve the complement levels as much as possible. No validation data is available to support these storage conditions. Thirty six normal samples and 56 clinical samples were tested on SPAPLUS™ and Tina-Quant C3c kit. Three of the clinical samples were diluted in SPAPLUS instrument system diluent to give adequate sample numbers over the measuring range. The samples were fairly distributed over the measuring range. The following results were obtained for 95 samples over the range of 0.230 to 3.670g/L:

Passing and Bablok fit $y = 0.99x + 0.00$
Linear regression $y = 0.9284 + 0.075$ $R^2 = 0.99$ $r = 0.993$

Scatter Plot with Passing &amp; Bablok Fit:


{9}

Residual Plot:

b. Matrix comparison:

Not applicable

# 3. Clinical studies:

a. Clinical sensitivity:

Not applicable

b. Clinical specificity:

Not applicable

c. Other clinical supportive data (when a. and b. are not applicable):

Not applicable

# 4. Clinical cut-off:

Not applicable

# 5. Expected values/Reference range:

These ranges were obtained using this kit, by measuring the C3c concentration of human serum. The reference interval was calculated using nonparametric statistics and represents the central $95\%$ of the population.

| | Number (n) | Mean (g/L) | Median (g/L) | 95 Percentile Range (g/L) |
| --- | --- | --- | --- | --- |
| C3c | 120 | 1.168 | 1.151 | 0.811 - 1.570 |

# N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

# O. Conclusion:

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