HUMAN CH50 REAGENT PACK FOR USE ON THE SPAPLUS, HUMAN CH50 CALIBRATOR SET FOR USE ON THE SPAPLUS, HUMAN CH50 CONTROLS FO

K113349 · The Binding Site Group , Ltd. · DAE · Jul 11, 2012 · Immunology

Device Facts

• Record ID: K113349
• Device Name: HUMAN CH50 REAGENT PACK FOR USE ON THE SPAPLUS, HUMAN CH50 CALIBRATOR SET FOR USE ON THE SPAPLUS, HUMAN CH50 CONTROLS FO
• Applicant: The Binding Site Group , Ltd.
• Product Code: DAE · Immunology
• Decision Date: Jul 11, 2012
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5240
• Device Class: Class 2

Indications for Use

These reagents are intended for the quantification of total classical complement activity (CH50) in human serum on the Binding Site SPA PLUS analyser. Measurement of complement activity aids in the diagnosis of immunological disorders, especially those associated with deficiencies of complement components. The test results are to be used in conjunction with clinical findings and other laboratory tests.

Device Story

The Human CH50 system consists of a reagent pack, calibrator set, and controls designed for use on the Binding Site SPAPLUS analyzer. The device measures total classical complement activity in human serum samples. The system is intended for use in clinical laboratory settings by trained personnel. Results are used by clinicians alongside other diagnostic tests to identify immunological disorders and complement deficiencies. The system automates the quantification process, providing objective data to support clinical decision-making regarding patient immune status.

Clinical Evidence

Clinical study evaluated 91 samples: 29 healthy controls and 62 CH50-deficient patients (autoimmune, kidney disease, and other pathologies). All 62 deficient samples were correctly identified as <41.6 U/mL, and all 29 healthy samples were >41.6 U/mL. Sensitivity: 100% (95% CI: 100-100%); Specificity: 100% (95% CI: 100-100%). Method comparison with predicate (n=110) showed Passing-Bablok regression y=1.15x-4.29, r=0.961, with 99.1% overall agreement.

Technological Characteristics

Turbidimetric assay. Reagents: liposomes with G6PDH, anti-DNP antibody (goat), G6P, NAD, maleate buffer. Measuring range: 12-95 U/mL. Instrumentation: SPA PLUS analyzer. Calibrators/controls: lyophilized, 6-level calibrator set, 3-level controls. Stability: 7 months unopened at 2-8°C; 30 days on-board. Analytical performance validated per CLSI EP-5A2, EP-6A, and EP-17A.

Indications for Use

Indicated for the quantification of total classical complement activity (CH50) in human serum to aid in the diagnosis of immunological disorders, particularly those associated with complement component deficiencies. Intended for use in adult populations.

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)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
k113349

B. Purpose for Submission:
New device

C. Measurand:
CH50, total complement activity

D. Type of Test:
Quantitative, turbidometric

E. Applicant:
The Binding Site Group, Ltd.

F. Proprietary and Established Names:
Human CH50 reagent pack for use on the SPA PLUS, Human CH50 calibrator set for use on the SPA PLUS, Human CH50 controls for use on the SPA PLUS

G. Regulatory Information:

1. Regulation section:
21 CFR § 866.5240 Complement components immunological test system
21 CFR § 862.1150 Calibrator
21 CFR § 862.1660 Quality Control Material (Assayed and Unassayed) –

2. Classification:
Class II, Device and Calibrator
Class I, Quality Control Material

3. Product codes:
DAE, Complement 9, Antigen, Antisera, Control

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JIX, Calibrator, Multi-Analyte Mixture

JJY, Multi-analyte controls, All kinds (Assayed and Unassayed)

4. Panel:
Immunology (82)
Chemistry (75)

H. Intended Use:

1. Intended use(s):
Human CH50 reagent pack for use on the SPA PLUS:
These reagents are intended for the quantification of total classical complement activity (CH50) in human serum on the Binding Site SPA PLUS analyser.
Measurement of complement activity aids in the diagnosis of immunological disorders, especially those associated with deficiencies of complement components. The test results are to be used in conjunction with clinical findings and other laboratory tests.

Human CH50 calibrator set for use on the SPA PLUS:
The Human CH50 calibrator set is intended for use on the SPA PLUS in conjunction with the Binding Site Human CH50 reagent pack (product code: NK095.S) for the determination of total complement activity.

Human CH50 controls for use on the SPA PLUS:
The Human CH50 controls are intended for use in conjunction with the Binding Site Human CH50 reagent pack (product code: NK095.S).

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

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

4. Special instrument requirements:
The Binding Site SPAplus™

I. Device Description:
The device consists of the following:
CH50 reagent pack includes: CH50 Liposome reagent R1(Liquid reagent containing

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G6PDH); CH50 Substrate R2 (lyophilized, containing anti-DNP antibody (goat), 24 mmol/L G6P and 9 mmol/L NAD); CH50 Substrate diluent R2a (Liquid reagent containing 10 mmol/L maleate buffer pH 5.0).

The CH50 calibrator set includes: Six levels, lyophilized.

The CH50 controls includes 3 level controls (low, high and elevated), 4x0.45 mL for each level, lyophilized.

# J. Substantial Equivalence Information:

1. Predicate device name(s) and K number(s):

WAKO Autokit CH50, WAKO CH50 Calibrator, WAKO Complement Control; k954145

2. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Binding Site Human CH50 reagent pack | WAKO Autokit CH50 |
| Intended Use: | Measurement of total complement activity | Same |
| Sample matrix | Serum | Same |
| Detection Method | Turbidimetric, absorbance | Same |
| Reagents | CH50 liposome reagent, substrate and substrate diluent | Same |
| CH50 anti-DNP antibody substrate mammalian source | Goat | Same |
| Differences | | |
| --- | --- | --- |
| Item | Binding Site Human CH50 reagent pack | WAKO Autokit CH50 |
| Instruments | SPA PLUS analyser | WAKO-30R analyzer |
| Measuring range | 12-95 U/mL | 10-60 U/mL |
| Expected values | 41.68-95.06 U/mL | 23-46 U/mL |

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| Similarities | | |
| --- | --- | --- |
| Item | Binding Site Human CH50 calibrator set and controls | WAKO CH50 Calibrators and Complement Controls |
| Reagents | Lyophilized | Same |
| Differences | | |
| --- | --- | --- |
| Item | Binding Site Human CH50 calibrator set and controls | WAKO CH50 Calibrators and Complement Controls |
| Traceability | Value assigned using Internal Reference which is compared to the predicate Internal Reference | Internal reference value assignment and determination per Mayer et al in Complement and complement fixation 1967 2nd ed. Springfield, Charles C. Thomas text book |
| Calibrator stability after reconstitution | Store on ice or 2-8°C and use within 10 hours | Store on ice and use within a day |
| Control stability after reconstitution | Store on ice or 2-8°C and use within 10 hours | Store on ice and use within 8 hours |

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

CLSI EP-5A2: Evaluation of Precision Performance of Clinical Chemistry; Approved Guideline - Second Edition

CLSI EP-6A: Evaluation of Linearity of Quantitative Measurements; Approved Guideline

CLSI EP-17A: Determinations of Limits of Detection and Limits of Quantitation; Approved Guideline

# L. Test Principle:

Liposomes encapsulating glucose-6-phosphate dehydrogenase (G6PDH) are used to mimic an invading microorganism. On addition of sample, antibodies in the reagent combine with dinitrophenyl groups on the surface of the liposomes. The resultant complex activates complement in the sample which lyses the liposome, releasing the

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G6PDH to react with glucose-6-phosphate and NAD in the reagent. The change in absorbance can be measured and is proportional to the complement activity in the sample. Comparison to a calibration curve gives a value for the unknown patient sample.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

The intra-assay and inter-assay precision was determined by testing four serum samples over 21 days with two runs per day on three different reagent lots. Results are summarized below.

| Sample | Mean (U/mL) | Within-Run | | Between-Run | | Between-Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV % | SD | CV % | SD | CV % | SD | CV % |
| Serum 1 | 78.73 | 1.67 | 2.1 | 3.04 | 3.90 | 3.26 | 4.1 | 4.76 | 6.0 |
| Serum 2 | 55.22 | 0.66 | 1.2 | 0.65 | 1.2 | 1.86 | 3.4 | 2.08 | 3.8 |
| Serum 3 | 46.05 | 0.84 | 1.8 | 1.03 | 2.2 | 1.00 | 2.2 | 1.66 | 3.6 |
| Serum 4 | 25.34 | 0.34 | 1.4 | 0.39 | 1.5 | 0.99 | 3.9 | 1.12 | 4.4 |

#### b. Linearity/assay reportable range:

Linearity across the assay range (12.68 – 95.47 U/mL) was confirmed by testing a serum pool with high range concentrations up to 95.9 U/mL. The samples were serially diluted 11 times with buffer (1:10) down to the lower measuring range (12.41 U/mL). All testing were performed three times. The regression plot equations where y is the measured level of CH50 concentration and x the theoretical concentration is as follows: y=0.996x – 2.709 (U/mL). r=0.994

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

Value assigned to the new device internal reference standard was compared to the predicate internal reference (IR) standard which was determined according to methods used by Mayer et al as described in Complement and complement fixation text book published in 1967 2nd ed. Springfield by Charles C. Thomas). The IR standard is used to assign values to the 6 calibrators and 3 controls.

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**Stability:**

Stability studies demonstrated the following claims:

- Unopened reagent pack, calibrators and controls: 7 months at 2-8°C
- On-board reagent pack: 30 days at 2-8°C
- Reconstituted calibrator and controls: 10 hours stored on ice or at 2-8°C

**d. Detection limit:**

The detection limits were determined by testing 60 replicates of a blank sample, the lowest calibrator, and a sample with value close to the blank sample. The limit of blank claim for this assay is 0.272 U/mL as determined by testing 60 replicates of a blank sample. The limit of detection represents the lowest measurable analyte level that can be distinguished from zero and has been estimated at 1.654 U/mL. The limit of quantitation is defined as the lowest amount of analyte that can be quantitatively determined and has been estimated as 11/98 U/mL for this assay.

**e. Analytical specificity:**

Interference by endogenous and other substances:

No significant assay interference was demonstrated by 1500 formazine turbidity units (FTU) of chyle, 200 mg/L bilirubin, or 5 g/L hemoglobin and 0.5 g/L ascorbic acid using CH50 sample at 36.26 U/mL.

The package insert states that “turbidimetric assays are not suitable for measurement of highly lipemic or hemolyzed samples, or samples containing high levels of circulating immune complexes due to the unpredictable degree of non-specific scatter these sample types might generate. Unexpected results should be confirmed using alternative assay method”.

**f. Assay cut-off:**

The cut-off of 41.68 U/mL for the Binding Site Human CH50 assay was established from 120 serum samples from normal healthy adult blood donors. The CH50 results ranged from 36.295 to 98.210 U/mL. A non-parametric distribution of CH50 results was seen that gave a 95 percentile reference lower limit of 41.68 U/mL.

**2. Comparison studies:**

**a. Method comparison with predicate device:**

A method comparison study between Human CH50 reagent pack, calibrator and controls on a SPA PLUS™ analyzer and predicate device was performed using 110 samples (29 normal, 62 known suppressed CH50 samples and 19 moderate level mixed samples to obtain results between 14-59 U/mL (5 samples were between 14-21 U/mL and 14 samples were between 32-59 U/mL). A total of 62 samples with results “&lt;13.88 U/mL*” results were

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excluded from the regression analysis. The study demonstrated the following Passing-Bablok fit: $y = 1.15x - 4.29$ (U/mL) with a correlation coefficient (r) of 0.961. ( $*&lt; 13.88$ U/mL is the lowest point of the calibration curve of calibrator used.)

Percent agreements between CH50 Reagent pack, calibrator and controls on a $\mathrm{SPAPLUS}^{\mathrm{TM}}$ analyzer and predicate in identifying CH50 deficient samples were calculated and summarized in the table below.

| | WAKO Autokit CH50 | | | |
| --- | --- | --- | --- | --- |
| | | Normal | CH50 deficient | Total |
| Binding Site Human CH50 | Normal | 42 | 0 | 42 |
| | CH50 deficient | 1 | 67 | 68 |
| | Total | 43 | 67 | 110 |

Positive percent agreement: $97.7\%$ (42/43) (95% CI: 93.2-102.2%)

Negative percent agreement: $100\%$ (67/67) (95% CI: 100-100%)

Overall percent agreement: $99.1\%$ (109/110) (95% CI: 97.3-100.9%)

b. Matrix comparison:

Not applicable

# 3. Clinical studies:

a. Clinical Sensitivity and Specificity:

A clinical study evaluated 91 samples on this kit: 29 normal and 62 CH50 deficient clinical samples. Of the 62 CH50 deficient samples, 33 were from autoimmune disease patients (19 SLE, 10 RA, 3 Sjögren's Syndrome, 1 Raynaud's syndrome), 8 from kidney disease patients (3 nephrotic syndrome, 3 nephritis, 1 nephropathy, 1 proteinuria), 21 other pathological disorders (1 neutropenia, 1 myelodysplasia, 1 bi-clonal gammopathy, 1 urticaria, 1 face swelling, 2 stroke, 14 undetermined). The 62 CH50 deficient clinical samples all had CH50 levels below $41.6~\mathrm{U / mL}$

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| N=91 | Patient Group | | | |
| --- | --- | --- | --- | --- |
| | | CH50 Deficient Autoimmune (33), kidney (8) and other pathological disorders (21) | CH50 Sufficient Healthy controls | Total |
| Binding Site Human CH50 | <41.6 U/mL | 62 | 0 | 62 |
| | >41.6 U/mL | 0 | 29 | 29 |
| | Total | 62 | 29 | 91 |

Sensitivity: 100% (62/62) (95% CI: 100-100%)

Specificity: 100% (29/29) (95% CI: 100-100%)

b. Other clinical supportive data (when a. is not applicable):

Not applicable.

4. Clinical cut-off:

Same as assay cut-off.

5. Expected values/Reference range:

Adult reference range:

Adult reference range was assessed using a total of 120 serum samples from healthy adult UK blood donors. A non-parametric distribution of CH50 results was seen that gave a 95 percentile reference interval of 41.68-95.06 U/mL with a mean of 68.37 U/mL and a median of 68.70 U/mL.

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.