HUMAN ALPHA2-MACROGLOBULIN KIT FOR USE ON SPAPLUS

K140664 · The Binding Site Group , Ltd. · DEB · Apr 16, 2014 · Immunology

Device Facts

• Record ID: K140664
• Device Name: HUMAN ALPHA2-MACROGLOBULIN KIT FOR USE ON SPAPLUS
• Applicant: The Binding Site Group , Ltd.
• Product Code: DEB · Immunology
• Decision Date: Apr 16, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5620
• Device Class: Class 2

Indications for Use

This kit is designed for the quantitative in vitro measurement of alpha-2-Macroglobulin in human serum using the SPAPLUS turbidimetric analyser. Measurement of alpha 2-Macroglobulin may aid in the diagnosis of blood-clotting or clot lysis disorders. This test should be used in conjunction with other laboratory and clinical findings.

Device Story

The Human alpha-2-Macroglobulin kit is an in vitro diagnostic reagent system designed for use on the SPAPLUS turbidimetric analyzer. It measures alpha-2-Macroglobulin levels in human serum samples. The device utilizes turbidimetric analysis to quantify the protein concentration. The system is intended for use in clinical laboratory settings by trained laboratory personnel. Results are provided to clinicians to assist in the diagnosis of blood-clotting or clot lysis disorders. The output is used in conjunction with other clinical and laboratory findings to support patient management decisions.

Clinical Evidence

No clinical data provided. The submission relies on bench testing and performance validation for the quantitative measurement of alpha-2-Macroglobulin on the SPAPLUS analyzer.

Technological Characteristics

Turbidimetric immunoassay kit for use on the SPAPLUS analyzer. Measures alpha-2-Macroglobulin in human serum. Operates via light scattering/turbidimetry principles. Designed for professional clinical laboratory use.

Indications for Use

Indicated for the quantitative in vitro measurement of alpha-2-Macroglobulin in human serum to aid in the diagnosis of blood-clotting or clot lysis disorders. For prescription use only.

Regulatory Classification

Identification

An alpha-2-macroglobulin immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the alpha-2-macroglobulin (a serum protein) in plasma. Measurement of alpha-2-macroglobulin may aid in the diagnosis of blood-clotting or clot lysis disorders.

Special Controls

*Classification.* Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 866.9.

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
k140664

B. Purpose for Submission:
New assay

C. Measurand:
Human alpha-2- Macroglobulin

D. Type of Test:
Quantitative immunoturbidometric

E. Applicant:
The Binding Site Group, Ltd.

F. Proprietary and Established Names:
Human alpha-2-Macroglobulin kit for use on SPAPLUS

G. Regulatory Information:

1. Regulation section:
21 CFR §866.5620, Alpha-2-Macroglobulin Immunological Test System

2. Classification:
Class II

3. Product code:
DEB, Alpha-2-Macroglobulin, Antigen, Antiserum, Control

4. Panel:
Immunology (82)

H. Intended Use:

1. Intended use(s):
This kit is designed for the quantitative in vitro measurement of alpha-2-Macroglobulin in human serum using the $\mathrm{SPA}_{\mathrm{PLUS}}$ turbidimetric analyser. Measurement of alpha 2-

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Macroglobulin may aid in the diagnosis of blood-clotting or clot lysis disorders. This 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 (k040958)

I. Device Description:

The kit contains the following reagents:

1. $1 \times 100$ Tests Human $\alpha 2$ -Macroglobulin Antiserum $\mathrm{SPAPLUS}$ (monospecific in stabilized liquid form with sodium azide and preservatives)
2. $1 \times$ Human $\alpha 2$ -Macroglobulin $\mathrm{SPAPLUS}$ Calibrator set 1-6 (6 x 1.0mL)
3. $2 \times 1.5 \mathrm{~mL}$ Human $\alpha 2$ -Macroglobulin $\mathrm{SPAPLUS}$ High Control (pooled human serum in stabilized liquid form with sodium azide and preservatives)
4. $2 \times 1.5 \mathrm{~mL}$ Human $\alpha 2$ -Macroglobulin $\mathrm{SPAPLUS}$ Low Control (pooled human serum in stabilized liquid form with sodium azide and preservatives)
5. $1 \times 100$ Test $\alpha 2$ -Macroglobulin Reaction Buffer $\mathrm{SPAPLUS}$

J. Substantial Equivalence Information:

1. Predicate device name(s):

N Antisera to Human $\alpha_{2}$ -Macroglobulin test system (Siemens)

2. Predicate $510(\mathbf{k})$ number(s):

k053073

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Measurement of α2-Macroglobulin to aid in the diagnosis of blood clotting or blood lysis disorders. | Same |
| Measuring range | 0.2-6.4 g/L | Same |

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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Sample type | Serum | Serum & heparinized plasma |
| Method | Turbidimetry | Nephelometry |
| Analyzer | SPAPLUS | BNII |
| Antibody | Goat anti-human α2-Macroglobulin | Rabbit anti-human α2-Macroglobulin |
| Open vial stability | Three months at 2 to 8°C | Four weeks at 2 to 8°C |
| On board stability | 30 days | Minimum 5 days at 8 hours per day for 5mL vials. Minimum 3 days at 8 hours per day for 2mL vials. |
| Shelf life stability | 12 months at 2-8°C | Not stated |
| Reference Interval | 0.74–2.98 g/L | 1.3–3.0 g/L |

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

CLSI EP06-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach

1. CLSI EP05-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline - Second Edition
2. CLSI EP17-A Protocols for Determination of Limits of Detection and Limits of Quantitation, Approved Guideline
3. CLSI EP07-A2 Interference Testing in Clinical Chemistry, Approved Guideline
4. CLSI C28-A3: Defining, Establishing and Verifying Reference Intervals in a Clinical Laboratory

## L. Test Principle:

This test measures levels of α2-Macroglobulin by turbidimetric methods, which involves the reaction of α2-Macroglobulin in serum with specific antiserum resulting in the formation of insoluble antigen/antibody complexes. When light is passed through the suspension, only a portion of the light is transmitted and focused onto a photodiode by an optical lens system; the remainder is scattered by the immunocomplexes. The amount of transmitted light is indirectly proportional to the specific antigen concentration in the test sample. α2-Macroglobulin concentrations are automatically calculated by reference to a calibration curve stored within the instrument.

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

### 1. Analytical performance:

a. Precision/Reproducibility:

This study was conducted according to the recommendations contained in CLSI EP-05-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline - Second Edition. Five samples with α2-Macroglobulin concentrations covering the measuring range were tested on three SPAPLUS

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instruments and using three lots of assays over 21 days, with two runs per day in duplicate, for a total of 84 replicate measurements. The sponsor's pre-defined acceptance criteria are total precision must be $\leq 10\%$ for all levels, within precision $\leq 5\%$ , and between run and between day $\leq 8\%$ CV.

The between-day and between-lot results from the lowest concentration sample (0.34 g/L) exceeded the acceptance criteria of $< 10\%$ CV; however, given that this concentration is at the bottom of the measuring range and is 10-fold above the medical decision point of $2.98~\mathrm{g / L}$ , this imprecision is unlikely to have an effect on clinical decisions, and the overall precision is deemed acceptable.

Repeatability

| Sample | Mean (g/L) | Within-Run | | Between-Run | | Between-Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 | 0.34 | 0.005 | 1.6 | 0.014 | 4.1 | 0.045 | 13.3 | 0.047 | 14.0 |
| 2 | 2.29 | 0.024 | 1.0 | 0.049 | 2.1 | 0.092 | 4.0 | 0.107 | 4.7 |
| 3 | 3.45 | 0.034 | 1.0 | 0.076 | 2.2 | 0.149 | 4.3 | 0.171 | 4.9 |
| 4 | 4.19 | 0.055 | 1.3 | 0.099 | 2.4 | 0.178 | 4.3 | 0.211 | 5.0 |
| 5 | 5.42 | 0.086 | 1.6 | 0.131 | 2.4 | 0.259 | 4.8 | 0.303 | 5.6 |

Instrument and Lot Reproducibility

| Sample | Mean (g/L) | # Runs | # Days | Between-Instrument | | Between-Lot | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | CV% | SD | CV% |
| 1 | 0.34 | 84 | 21 | 0.017 | 4.94 | 0.040 | 11.82 |
| 2 | 2.29 | 84 | 21 | 0.076 | 3.33 | 0.048 | 2.09 |
| 3 | 3.45 | 84 | 21 | 0.107 | 3.08 | 0.031 | 0.89 |
| 4 | 4.19 | 84 | 21 | 0.121 | 2.89 | 0.095 | 2.26 |
| 5 | 5.42 | 84 | 21 | 0.187 | 3.45 | 0.161 | 2.98 |

# b. Linearity/assay reportable range:

i. The analytical measuring range of the assay is $0.2 - 6.4\mathrm{g / L}$
ii. Linearity

A high and a low pool were prepared for Human $\alpha 2$ -Macroglobulin kit for use on SPAPLUS in order to evaluate linearity across the measuring range. The pools were prepared for each assay. Normal human serum was depleted of $\alpha 2$ -macroglobulin by affinity chromatography. For the high pool, a healthy donor serum sample was spiked with a processed serum sample, preserved with citrate

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$\beta$ -alanine, that consisted of pooled healthy donor sera and disease state sera. The pools were combined in a single proportional dilution series (e.g., $90 + 10$ , $80 + 20$ , etc.), containing from $100\%$ high pool to $0\%$ high pool.

Three replicates of each level of the dilution series were run and the mean value calculated. All of the concentrations within the analytical measuring range met the sponsor's acceptance criterion of $\% \mathrm{CV} < 8\%$ and all concentrations met the sponsor's regression analysis acceptance criteria:

| Slope | r | %CV for each level | Allowable mean % recovery |
| --- | --- | --- | --- |
| 0.90 – 1.10 | ≥ 0.975 | <8% | <10% |
| % High pool | Mean Unweighted result (g/L) | Expected Unweighted Result (g/L) | % CV | Mean Recovery | Min | Max |
| --- | --- | --- | --- | --- | --- | --- |
| 100.0% | 6.667 | 6.555 | 1.5% | 103.45% | 101.70% | 104.60% |
| 90.0% | 5.897 | 5.897 | 2.0% | 101.71% | 99.42% | 103.32% |
| 80.0% | 5.241 | 5.239 | 2.8% | 101.74% | 98.49% | 103.54% |
| 70.0% | 4.567 | 4.581 | 0.9% | 101.41% | 100.42% | 102.20% |
| 60.0% | 3.863 | 3.922 | 1.2% | 100.16% | 98.99% | 101.37% |
| 50.0% | 3.142 | 3.264 | 1.8% | 97.87% | 95.85% | 99.19% |
| 40.0% | 2.370 | 2.606 | 2.9% | 92.48% | 90.90% | 95.58% |
| 30.0% | 1.823 | 1.948 | 1.1% | 95.15% | 94.19% | 96.28% |
| 20.0% | 1.275 | 1.290 | 1.0% | 100.42% | 99.66% | 101.55% |
| 10.0% | 0.658 | 0.631 | 1.1% | 105.76% | 104.43% | 106.51% |
| 5.0% | 0.302 | 0.302 | 2.3% | 101.12% | 98.66% | 103.35% |
| 4.0% | 0.246 | 0.237 | 1.1% | 104.99% | 104.14% | 106.27% |

Regression analysis

| | Slope (95% CI) | Intercept (95% CI) | r |
| --- | --- | --- | --- |
| α2-Macroglobulin | 1.01 (0.98–1.04) | -0.06 (-0.16–0.04) | 0.999 |

These data support linearity throughout the analytical measuring range of the assay $0.2 - 6.4\mathrm{g / L}$

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

a. Calibrators and Controls

Calibrator

The master calibrator is traceable to international reference standard DA470k.

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Calibrators are prepared from pooled human sera collected from individual subjects and shown to contain suitable concentrations of α2-Macroglobulin. The samples are pooled and processed to provide a stable fluid for use in turbidimetric assays. The assigned values of the calibrator set must be within ±15% of the target values to pass the sponsor’s acceptance criteria.

b. Controls

Controls are prepared from pooled human sera collected from individual subjects and shown to contain suitable concentrations of α2-Macroglobulin. The samples are pooled and processed to provide a stable fluid for use in turbidimetric assays. The final value assignment of the high and low controls is carried out by running five replicates of the control fluids on the previously generated five calibration curves. The mean result is multiplied by the same adjustment factor to give the final assigned value of the controls. A control assignment check is carried out by multiplying each of the control fluid results by the adjustment factor and calculating the percentage difference between the experimental and assigned value.

c. Stability

Shelf life: The sponsor provided data that supported the claim that the kits are stable for 12 months when stored unopened at 2-8°C.

d. On-board: The sponsor provided data that supported the claim that the kits are stable for up to 30 days when stored on-board the instrument provided the temperature is maintained at 8-12°C.

e. Open vial: The sponsor provided data that supported the claim that the kits are stable for 3 months when stored closed at 2-8°C.

ii. Antigen

The antiserum used in the Human α2-Macroglobulin kit for use on SPAPLUS is purchased from a commercial source. The anti-α2-Macroglobulin immunoglobulins are chromatographically concentrated to increase the specific antibody concentration of the α2-Macroglobulin antiserum. The antibodies are then characterized by immunoelectrophoresis (IEP) versus human serum.

d. Detection limit:

Analytical sensitivity was determined based on the recommendations contained in EP17-A Protocols for Determination of Limits of Detection and Limits of Quantitation, Approved Guideline.

i. Limit of Blank (LoB): The LoB sample consisted of an analyte-depleted sample pool. The LoB sample was tested 60 times and the mean and standard deviation (SD) were calculated.

LoB was calculated using non-parametric analysis. The LoB results were

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ranked from lowest to highest concentration, and the LoB was estimated to be the result equivalent to the concentration at the $95^{\text{th}}$ percentile, or between sample results 57 and 58. The LoB was determined to be $0.018 \, \text{g/L}$ .

ii. Limit of Detection (LoD): The LoD was calculated from the LoB and the combined SDs of the LoQ samples: $\mathrm{LoD} = \mathrm{LoB} + [(1.645 \times \mathrm{SDs})]$ . The LoD was determined to be $0.031 \mathrm{~g} / \mathrm{L}$ .

| Acceptance criterion | Result |
| --- | --- |
| LoD < LoQ | 0.0313 g/L < 0.20 g/L |
| TE < 0.074 g//L | 0.0212 g/L |

iii. Limit of Quantitation (LoQ): five US normal donor samples with known $\alpha 2$ -Macroglobulin concentrations were diluted with analyte-depleted serum so that the concentrations were close to the bottom of the standard measuring range $(0.19 - 0.21\mathrm{g / L})$ . The five samples were run 12 times over five days using the Human $\alpha 2$ -Macroglobulin Kit for use on $\mathrm{SPA}_{\mathrm{PLUS}}$ analyser. The samples were also tested in triplicate over the same time period on the predicate device (BN II analyser using N Antiserum to Human $\alpha 2$ -Macroglobulin reagents). LoQ was calculated according to the formula $\mathrm{TE} = |[\mathrm{mean~SPAPLUS~assay}] - [\mathrm{mean~predicate~assay}]| + 2\times \mathrm{SDs}$ . The LoQ was determined to be $0.20\mathrm{g / L}$ , which is at the bottom of the measuring range.

# e. Analytical specificity:

# i. Interference

This study was performed according to the recommendations contained in CLSI EP07-A2 Interference Testing in Clinical Chemistry, Approved Guideline, except that the cutoff for acceptable interference was set as $\mathrm{D_{obs}} &lt; \mathrm{D_{max}}$ and $&lt; 10\%$ difference between the mean of the spiked sample and the unspiked sample. Twenty replicates of each of the serum base pools were run in order to calculate standard deviation. The $\mathrm{D_{max}}$ was set at $10\%$ of the analyte concentration for each of the base pools. The standard deviation and the $\mathrm{D_{max}}$ were then used to calculate the number of replicates required in the interference study. Three replicates of each spiked and unspiked concentration were determined sufficient for this study.

Three (3) human serum base pools were prepared to target three analyte concentrations. These levels were chosen to evaluate the effects the interferents at critical clinical concentrations. The high concentration pool was diluted with analyte-free serum to achieve the desired target concentrations.

The serum pools were spiked separately with hemoglobin (5 g/L), bilirubin (200 mg/L), Intralipid (500 mg/dL) and triglyceride (1000 mg/dL). The spiked pools were analyzed, as were the unspiked pools, which were prepared by spiking the base pools separately with the same volume of commercially obtained blank for each potential interferent.

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Hemoglobin 5 g/L

| Interferent Hemoglobin | Mean blank result (g/L) | Mean interferent result (g/L) | % Difference | Dobs (g/L) | Dmax (g/L) | Pass/Fail |
| --- | --- | --- | --- | --- | --- | --- |
| Level 1 | 1.524 | 1.414 | -7.22% | -0.110 | ±0.174 | Pass |
| Level 2 | 2.541 | 2.495 | -1.81% | -0.046 | ±0.314 | Pass |
| Level 3 | 3.794 | 3.900 | 2.79% | 0.106 | ±0.426 | Pass |

Bilirubin 200 mg/L

| Interferent Bilirubin | Mean blank result (g/L) | Mean interferent result (g/L) | % Difference | Dobs (g/L) | Dmax (g/L) | Pass/Fail |
| --- | --- | --- | --- | --- | --- | --- |
| Level 1 | 1.711 | 1.689 | -1.29% | -0.022 | ±0.174 | Pass |
| Level 2 | 2.834 | 2.980 | 5.15% | 0.145 | ±0.314 | Pass |
| Level 3 | 4.161 | 4.105 | -1.35% | -0.057 | ±0.426 | Pass |

Intralipid 500 mg/dL

| Interferent Intralipid | Mean blank result (g/L) | Mean interferent result (g/L) | % Difference | Dobs (g/L) | Dmax (g/L) | Pass/Fail |
| --- | --- | --- | --- | --- | --- | --- |
| Level 1 | 1.287 | 1.332 | 3.522% | 0.045 | ±0.144 | Pass |
| Level 2 | 2.591 | 2.587 | -0.15% | -0.003 | ±0.292 | Pass |
| Level 3 | 2.989 | 2.968 | -0.70% | 0.009 | ±0.364 | Pass |

Triglyceride 1000 mg/dL

| Interferent Triglyceride | Mean blank result (g/L) | Mean interferent result (g/L) | % Difference | Dobs (g/L) | Dmax (g/L) | Pass/Fail |
| --- | --- | --- | --- | --- | --- | --- |
| Level 1 | 1.414 | 1.451 | 2.62% | 0.037 | ±0.154 | Pass |
| Level 2 | 2.720 | 2.752 | 1.18% | 0.033 | ±0.290 | Pass |
| Level 3 | 3.834 | 3.755 | -2.06% | -0.079 | ±0.394 | Pass |

These results demonstrate that no clinically significant interference was observed at the indicated concentrations.

ii. Hook effect

The sponsor provided data demonstrating that there is no hook effect at concentrations of antigen up to 12.8 g/L.

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f. Assay cut-off:

Same as clinical cutoff; see below.

2. Comparison studies:

a. Method comparison with predicate device:

154 samples across the analytical measuring range of the assay (see below for demographics and sample distribution) were tested on the predicate method and on the $\alpha$-2-Macroglobulin kit for use on SPAPLUS. Samples from subjects with diabetes, alcoholic liver disease, high levels of CRP and amyloid disease were included, as well as normal samples to ensure the measuring range was covered. Ten high concentration samples were constructed by spiking normal serum with purified $\alpha$-2-macroglobulin. Linear, Passing-Bablock, and Deming regression analyses were performed and no outliers were removed.

| Presenting Symptom or Admission Diagnosis | Number of Samples |
| --- | --- |
| UK Asian Diabetic | 24 |
| Amyloid | 17 |
| High CRP | 19 |
| Unknown | 19 |
| Alcoholic liver disease | 23 |
| Normal Human Serum | 42 |
| Constructed samples- normal human serum with purified α2-Macroglobulin | 10 |
| Total | 154 |

The sponsor's predetermined acceptance criterion was that substantial equivalence would be demonstrated if the results were within $\pm 10\%$ Passing-Bablock slope bias, and each individual result obtained on the test device should be within $\pm 20\%$ of the result obtained for that sample on the predicate device. Sample demographics: 154 samples, age range 19-93 years, $33\%$ male, $38\%$ female, and $29\%$ unknown.

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| Sample Distribution | |
| --- | --- |
| N | 154 |
| Concentration Range | 0.696–6.072 g/L |
| < 25% of cutoff | N =53 |
| Cutoff ± 25% (2.98 g/L) | N= 63 |
| >25% of cutoff | N= 34 |

Regression Results:

| Regression Analysis | Slope 95% CI | Intercept 95% CI |
| --- | --- | --- |
| Passing- Bablok y= 1.00x – 0.05 | 0.97 to 1.04 | -0.13 to 0.03 |
| Weighted Deming y= 1.01x – 0.07 | 0.98 to 1.05 | -0.13 to 0.00 |
| Linear regression y=0.98x + 0.03
R= 0.977 | 0.94 to 1.01 | -0.08 to 0.14 |

b. Matrix comparison:
Not applicable

3. Clinical studies:
a. Clinical Sensitivity and specificity:
Not applicable
b. Other clinical supportive data (when a. is not applicable):
Not applicable

4. Clinical cut-off:
The clinical decision point is set to 2.98 g/L, which is the top of the 95th percentile of the reference range for healthy individuals as determined in the reference range study, below.

5. Expected values/Reference range:
A reference range study was performed according to CLSI C28-A3: Defining, Establishing and Verifying Reference Intervals in a Clinical Laboratory. 166 normal healthy samples from US and German donors that were not used for the Method Comparison study were evaluated.

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| Results | |
| --- | --- |
| N | 166 |
| Range | 0.390–3.739 g/L |
| Mean | 1.580g/L |
| Male | 108 (65%) |
| Median | 1.500g/L |
| 95^{th} percentile | 0.74–2.98g/L |
| 95% CI lower boundary | 0.6765– 0.8134 |
| 95% CI upper boundary | 2.7157–3.2655 |

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.

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