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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:

K173704

B. Purpose for Submission:

Modification to an existing device

C. Measurand:

Tryptase

D. Type of Test:

Fluorescence immunoassay

E. Applicant:

Phadia US Inc.

F. Proprietary and Established Names:

ImmunoCAP Tryptase

ImmunoCAP Tryptase Calibrators

ImmunoCAP Tryptase Conjugate 50

ImmunoCAP Tryptase Calibrator Strip, ImmunoCAP Tryptase Control Strip

G. Regulatory Information:

1. Regulation section:

21 CFR §866.5760, Tryptase test system

2. Classification:

Class II

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3. Product code:

OYL; Tryptase Assay System

4. Panel:

Immunology (82)

H. Intended Use:

1. Intended use:

ImmunoCAP Tryptase is an in vitro semi-quantitative assay for measurement of tryptase in human serum or plasma (EDTA, lithium heparin or sodium heparin). It is intended for in vitro diagnostic use as an aid in the clinical diagnosis of patients with a suspicion of systemic mastocytosis in conjunction with other clinical and laboratory findings. ImmunoCAP Tryptase is to be used with the instruments Phadia 100, and Phadia 250.

2. Indication for use:

Same as intended use.

3. Special conditions for use statement:

Prescription use only.

4. Special instrument requirements:

For use with the Phadia 100 and Phadia 200 instruments

I. Device Description:

ImmunoCAP Tryptase is a fluorescence immunoassay for the measurement of total tryptase in human serum or plasma, based on the ImmunoCAP solid phase. ImmunoCAP Tryptase concentrations are semi-quantitatively reported in microgram/L (μg/L). ImmunoCAP Tryptase reagents are modular in concept and are available individually in different package sizes dependent on instrument system used.

J. Substantial Equivalence Information:

1. Predicate device name:

ImmunoCAP Tryptase

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2. Predicate 510(k) number:

K103039

3. Comparison with predicate:

Similarities Item Device Predicate

Intended Use ImmunoCAP Tryptase is an in vitro semi-quantitative assay for measurement of tryptase in human serum or plasma (EDTA, lithium heparin or sodium heparin). It is intended for in vitro diagnostic use as an aid in the clinical diagnosis of patients with a suspicion of systemic mastocytosis in conjunction with other clinical and laboratory findings. ImmunoCAP Tryptase is to be used with the instruments Phadia 100 and Phadia 250

ImmunoCAP Tryptase is an in vitro semi-quantitative assay for measurement of tryptase in human serum or plasma (EDTA, lithium heparin or sodium heparin). It is intended for in vitro diagnostic use as an aid in the clinical diagnosis of patients with a suspicion of systemic mastocytosis in conjunction with other clinical and laboratory findings. ImmunoCAP Tryptase is to be used with the instruments Phadia 100, Phadia 250 and Phadia 1000.

Sample matrix Human serum or plasma (EDTA, lithium heparin or sodium heparin)

Same

Tryptase Conjugate Anti-tryptase mouse monoclonal antibodies coupled to ß- Galactosidase

Same

Analytical Technology Immunofluorescence measurement

Same

Reporting of Results Semi-quantitative, μg/L Same Analytical sensitivity (LoD)

< 1 μg/L Same

Software Same ImmunoCAP Tryptase method on IDM and Prime (data management software)

Same

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Differences Item Device Predicate

Assay system (Tryptase Anti-Tryptase, Tryptase Control, Tryptase Sample Diluent, Instruments)

ImmunoCAP Tryptase run on Phadia 100 and Phadia 250.

ImmunoCAP Tryptase run on Phadia 100, Phadia 250 and Phadia 1000.

Tryptase Conjugate raw material

Fragmented F(ab’)2 antibody

Concentration ß-Galactosidase-anti-tryptase: approximately 1.6 μg/ml

Intact antibody

Concentration ß-Galactosidase-anti- tryptase: approximately 3.5 μg/ml

Tryptase Calibrators/Curve control raw material

Human recombinant tryptase

Human lung tryptase

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

CLSI EP25-A: Evaluation of Stability of In Vitro Diagnostic reagents; Approved Guideline.

CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline-Third Edition.

CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline-Second Edition.

CLSI EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline Second Edition.

L. Test Principle:

Anti-tryptase, covalently coupled to ImmunoCAP, reacts with tryptase in the patient sample. After washing, enzyme labeled antibodies against tryptase are added to form a complex. After incubation, unbound enzyme-anti-tryptase is washed away and the bound complex is then incubated with a developing agent. After stopping the reaction, the fluorescence of the eluate is measured. The higher the response value, the more tryptase is present in the sample. To evaluate the test results, the responses of the patient samples are transformed into concentrations with the use of a calibration curve.

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M. Performance Characteristics:

1. Analytical performance:

All results below fulfilled the manufacturer’s pre-determined acceptance criteria.

a. Precision/Reproducibility:

Phadia 100

Eighteen serum samples with tryptase levels covering the Analytical Measuring Range (AMR) were assayed on three different Phadia 100 instruments. Samples were run in replicates of four on seven different days within a period of 21 days. The results of the study are displayed in the following table:

Sample N Mean (ug/L)

Within-Run

(%CV)

Between-Run

(%CV)

Between-Instrument

(%CV)

Total (%CV)

1 84 1.9 2.9 2.0 2.8 4.5 2 84 2.8 2.6 2.3 2.6 4.4 3 84 3.3 2.5 2.2 3.1 4.6 4 84 4.0 1.6 3.9 0.5 4.3 5 84 5.0 1.8 1.6 2.3 3.4 6 84 7.3 1.9 2.3 1.1 3.1 7 84 10.4 1.5 1.7 1.0 2.4 8 84 10.6 1.9 3.1 1.0 3.8 9 84 17.9 1.7 2.0 0.0 2.6 10 84 19.6 1.9 2.1 0.0 2.9 11 84 20.5 2.7 2.2 0.0 3.5 12 84 35.7 1.6 3.2 0.0 3.6 13 84 36.7 3.4 3.1 0.0 4.6 14 84 60.8 1.8 2.9 0.0 3.4 15 84 137.8 2.2 3.3 0.0 4.0 16 84 151.2 2.6 2.8 1.0 3.9 17 84 167.1 2.3 3.3 0.0 4.0 18 84 168.1 2.6 3.5 0.6 4.4

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Phadia 250

Twenty one serum samples with different tryptase levels covering the AMR were assayed on three different Phadia 250 instruments. The results of the study are displayed in the following table:

Sample N Mean (ug/L)

Within-Run

(%CV)

Between-Run

(%CV)

Between-Instrument

(%CV)

Total (%CV)

1 84 1.7 2.3 5.6 3.2 6.9 2 84 2.3 2.4 5.2 2.2 6.1 3 84 2.7 2.7 5.2 1.7 6.1 4 84 3.5 3.4 5.8 0.0 6.7 5 84 3.6 2.3 4.4 1.1 5.1 6 84 6.8 1.7 4.4 0.0 4.7 7 84 10.3 2.6 4.7 1.0 5.4 8 84 11.9 2.1 3.5 1.3 4.3 9 84 16.8 2.3 5.1 1.0 5.7 10 84 18.1 1.6 4.3 1.1 4.7 11 84 19.8 2.2 4.6 0.0 5.1 12 84 22.3 2.1 4.5 2.4 5.5 13 84 36.6 2.1 4.5 1.1 5.1 14 84 63.2 2.2 5.7 2.9 6.7 15 84 76.3 2.4 6.1 1.1 6.7 16 84 115.9 3.1 6.3 3.3 7.7 17 84 122.7 3.4 7.1 2.4 8.2 18 84 133.6 3.3 7.2 3.9 8.8 19 84 161.0 4.6 6.9 2.7 8.8 20 84 168.1 4.1 7.4 0.7 8.5 21 84 171.4 4.9 6.3 0.0 8.0

Lot-to-lot Reproducibililty

Ten serum samples with tryptase concentrations covering the AMR of the assay were assayed using three different lots of reagent that included the updated ImmunoCAP Tryptase Conjugate, the updated ImmunoCAP Tryptase Calibrators and the

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ImmunoCAP Tryptase Anti-Tryptase. The conjugate, calibrators and anti-Tryptase lots were combined into six different combinations. Each sample was tested in replicates of three, with each of the six combinations in three assay runs, for a total of 54 measurements per sample. The results of the study are displayed in the following table:

Sample N Mean (µg/L)

Within-run

(%CV)

Between -lot

(%CV)

Between-run

(%CV)

Total (%CV)

1 54 2.8 4.6 4.5 0.0 6.4 2 54 3.4 3.5 2.5 0.0 4.3 3 54 7.5 3.1 1.8 1.9 4.0 4 54 10.5 2.5 2.5 1.9 4.0 5 54 16.9 2.7 1.3 1.1 3.2 6 54 19.4 2.8 2.4 1.4 4.0 7 54 33.6 3.6 1.0 3.1 4.8 8 54 136.6 4.8 4.1 4.0 7.5 9 54 159.4 5.5 3.4 0.8 6.5 10 54 162.0 5.8 1.3 4.0 7.2

b. Linearity/assay reportable range:

Three serum samples with tryptase concentrations between 160 – 200 μg/L were diluted in 2-fold direct dilutions in the ImmunoCAP IgE/ECP/Tryptase sample diluent. Samples (undiluted and diluted) were analysed in replicates of four in one assay run on the Phadia 100 and Phadia 250 instruments.

Phadia 100 Regression Analysis Sample Slope (95% CI) Y-intercept (95% CI) r2

1 0.99 (0.97–1.02) −0.75 (−2.62–1.11) 0.9993 2 1.02 (0.97–1.06) 0.22 (0.97–1.06) 0.9981 3 0.99 (0.96–1.03) −1.23 (0.96–1.03) 0.9987

Phadia 250 Regression Analysis Sample Slope (95% CI) Y-intercept (95% CI) r2

1 0.99 (0.95–1.03) −1.11 (−3.97–1.75) 0.9983 2 1.01 (0.97–1.06) 0.76 (−2.85–4.38) 0.9981 3 1.00 (0.98–1.01) −0.38 (−1.32–0.57) 0.9998

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

Traceability There is no internal standard for tryptase. ImmunoCAP Tryptase calibrators and ImmunoCAP Tryptase Curve Control is prepared from recombinant tryptase protein. The recombinant tryptase is expressed in Sf9 insect cells infected with recombinant baculovirus coding for mast-cell tryptase. The original reference standard was produced in-house. New in-house reference standard is produced from the stock solutions approximately every 3rd year. Each subsequent lot is standardized against the original reference standard.

Stability Calibrator stability: Calibrator stability was evaluated using three different lots. For the calibrator strip, one lot of ImmunoCAP Tryptase Calibrator strip was first transport simulated and then stored at 32ºC for 28 days to evaluate on-board reagent stability. Following this, time zero measurement with two additional calibroator lots were performed. All three calibrator lots were stored at 2°–8°C. The three lots were tested at five different test occasions up to 25 months. The results support an on-board stability claim of 28 days and a real-time stability claim of 25 months when calibrators are stored at 2–8ºC.

Conjugate Stability: To simulate transport stability, one lot of ImmunoCAP Tryptase Conjugate 50 was stored at 32ºC for one week. During this time the conjugates were moved to 2–8ºC twice for 18–24 hours and moved once to −20ºC for 2–3 hours. Following transport simulation, the conjugate was left on board for 4 days at 2–8ºC, and were recapped every night. After the initial time zero measurements, all three lots were stored at 2–8ºC and stability was measured at four additional time points up to 25 months. The results support an onboard stability claim of four days when stored on the Phadia 250, and real-time stability claim of 25 months when stored at 2-8ºC.

d. Detection limit:

The LoB was determined using five tryptase free serum sample. Samples were tested in replicates of four, in three separate runs, during three days, with two different lots of reagents (ImmunoCAP Tryptase Conjugate, ImmunoCAP, Tryptase Calibrators and ImmunoCAP Tryptase Anti-Tryptase) for a total 60 blank determinations. The LoB was estimated from Relative Units (RU) as the maximum value for reagent lot-specific LoB estimates. The LoB was determined to be less than 1 µg/L.

The LoD was determined by measuring five low positive samples ranging from LoB to 5x LoB. Also, the lowest tryptase calibrator (1 µg/L) was assayed as a sample. Each sample was assayed in replicates of four, in three separate runs, for three days, with two different lots of reagents (ImmunoCAP Tryptase Conjugate, ImmunoCAP Tryptase Calibrators and ImmunoCAP Tryptase Anti-Tryptase) for a total of 60 low

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positive determinations. Calculation of LoD was made on Response Units (RU) because no tryptase concentrations are calculated below the lowest calibrator. The LoD was determined to be less than 1 µg/L (1µg/L is the lowest calibrator in the kit). The data is presented in the table below:

Instrument Mean

Calibrator 1 (RU)

LoB (RU)

LoD (RU)

Phadia 100 250 89 116

Phadia 250 284 83 109

e. Analytical specificity:

Studies were performed in alignment with CLSI EP07-A2: Interference Testing in Clinical Chemistry; Approved Guideline ‐ Second Edition. Three serum samples with different concentrations of tryptase; approximately 4 μg/L, 18 μg/L and 140 μg/L were used. Serum samples were spiked with five different concentrations of potentially interfering substances. Bilirubin F (up to 19.4 mg/dl), Bilirubin C (up to 20.3 mg/dl), Hemoglobin (up to 490 mg/dl), Chyle (up to 7160 FTU), Rheumatoid Factor (up to 500 IU/ml), and Heparin (up to 75 U/ml) were used to spike serum samples. For reference, the serum samples were diluted with the same volume of buffer solution (unspiked). For Heparin, the untreated serum samples were used as reference (unspiked). The samples were analyzed in 3 replicates in one assay run. No significant interference at the tested levels was observed.

f. Assay cut-off:

The cut-off of 20 μg/L of tryptase is internationally recognized and is designated by the World Health Organization (WHO) as a minor criterion in the diagnosis of systemic mastocytosis. The WHO classification for systemic mastocytosis is described in the “WHO classification of tumours of haematopoietic and lymphoid tissues” published in 2008.

g. Hook-effect:

Hook effect was evaluated by comparing the response of a serum sample with a tryptase concentration of 2000 μg/L with the response of the highest calibrator, which is at concentration of 200 μg/L, and ensuring that the response level of the high sample was clearly above the highest calibrator. The hook sample and the highest calibrator were assayed in 24 replicates respectively in one assay run in the Phadia 100 and 250 instruments. No hook effect was observed.

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2. Comparison studies:

a. Method comparison with predicate device:

Comparison between the updated ImmunoCAP Tryptase and predicate ImmunoCAP Tryptase (K103039) was performed with 275 serum samples with tryptase levels across the analytical measuring range of the the device. Passing-Bablock regression analysis and 95% confidence intervals (CI) for the intercept and slope were estimated for all samples and all samples excluding healthy individuals.

Comparison Slope (95% CI) y-intercept (95% CI) All 0.948 (0.94–0.95) 0.418 (0.35–0.48)

Excluding Healthy Individuals 0.959 (0.94–0.98) −0.13 (−0.06–0.25)

b. Instrument comparison between Phadia 100 and Phadia 250:

Conformity between the Phadia 250 and Phadia 100 instruments for the updated ImmunoCAP Tryptase using 69 serum samples with tryptase concentrations covering the measuring range. Samples were assayed in one replicate in two assay runs on three different Phadia 100 and three different Phadia 250 instruments, for six runs/instrument system. Passing-Bablok regression analysis was used to evaluate the bias between tryptase measurements from both instruments. The results are presented in the table below:

Comparison Slope (95% CI) y-intercept (95% CI) Phadia 250 vs Phadia

100 0.92 (0.91–0.94) 0.12 (-0.07–0.57)

c. Matrix comparison:

Twenty-five patient samples containing different levels of tryptase ranging from approximately 4 –190 μg/L, were used in the study. The matrices compared were: serum, EDTA plasma, lithium heparin plasma and sodium heparin plasma. All samples matrices from each patient were tested in replicates of two in one assay run. Passing-Bablok regression analysis was used to analyze data. The results are presented in the table below:

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Comparison Slope (95% CI) Intercept (95% CI)

EDTA vs. Li Heparin 1.02 (0.95–1.05) -0.48 (-1.37–0.33) EDTA vs. Na Heparin 0.98 (0.95–1.02 0.15 (-0.79–0.63)

EDTA vs Serum 0.98 (0.92–1.05) -0.02 (-0.51–2.19) Li Heparin vs Na Heparin 0.97 (0.93–1.01) 0.48 (-0.30–1.11)

Li Heparin vs Serum 0.98 (0.93–1.03) 0.44 (-0.27–1.87) Na Heparin vs Serum 1.00 (0.94–1.06) 0.21 (-0.71–1.61)

3. Clinical studies:

a. Clinical Sensitivity and Specificity:

Clinical performance of the ImmunoCAP Tryptase assay was evaluated at two independent sites:

Site 1:

The study was performed with samples collected from 84 patients with a suspicion of mastocytosis. 77 subjects were adults ( >22 years of age) and 7 subjects were pediatric patients (<22 years of age). All patients were referred to the site due to a suspicion of mastocytosis and other mast cell-related diseases on the basis of clinical symptoms attributable to mast cell activation (MCA) including anaphylaxis. Patients were classified with and without the WHO recommended fourth minor criterion of tryptase levels persistently exceeding 20 μg/L.

WHO Classification including tryptase

+ - Total ImmunoCAP

Tryptase Assay (cut-off > 20

μg/L )

+ 37 4 41 - 19 24 43

Total 56 28 84

Sensitivity = 66.1% (95% CI: 52.2–78.2) Specificity = 85.7% (95% CI: 67.3–96.0 PPV = 90.2% (95% CI: 76.9–96.0) NPV = 55.8% (95% CI: 39.9–70.9)

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WHO Classification excluding tryptase

+ - Total ImmunoCAP

Tryptase Assay (cut-off > 20

μg/L )

+ 32 9 41 - 19 24 43

Total 51 33 84

Sensitivity = 62.7% (95% CI: 48.1–75.9) Specidficity = 72.7% (95% CI: 54.5–86.7) PPV = 78.0% (95% CI: 62.4–89.4) NPV = 55.8% (39.9–70.9)

Site 2: The study was performed with samples from 54 patients with a suspicion of mastocytosis. 42 subjects were adults (≥ 22 years of age) and 12 were pediatric patients (< 22 years of age). All cases were consecutively referred due to the suspicion of mastocytosis or mast cell activation syndrome based primarily on clinical symptoms attributed to MCA, including anaphylaxis, a characteristic skin lesion or lesional skin biopsy, a bone marrow biopsy or an elevated baseline serum tryptase level. Patients were classified with and without the WHO recommended fourth minor criterion of tryptase levels persistently exceeding 20 μg/L.

WHO Classification including tryptase

+ - Total ImmunoCAP

Tryptase Assay (cut-off > 20

μg/L )

+ 15 3 18 - 10 26 36

Total 25 29 54

Sensitivity = 76.2% (95% CI: 52.8–91.8) Specificity = 66.7.% (95% CI: 48.2–82.0) PPV = 59.3% (95% CI: 76.938.8–77.6) NPV = 81.5% ( 95% CI: 61.9–93.7)

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WHO Classification excluding tryptase

+ - Total ImmunoCAP

Tryptase Assay (cut-off > 20

μg/L )

+ 15 3 18 - 10 26 36

Total 25 29 54

Sensitivity = 75.0% (95% CI: 50.9–91.3) Specificity = 64.7% (95% CI: 46.5–80.3) PPV = 55.6% (95% CI: 35.3–74.5) NPV = 81.5% (95% CI: 61.9–93.7)

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:

Serum samples from 163 healthy individuals between 1–68 years of age were analyzed in duplicates on both the Phaida 100 and Phadia 250 instruments. Of these 70 were males 0–68 years of age (median age 29), and 93 were females 1-–68 years of age (median age 40). Pediatric samples (below the age of 22) comprised 68 individuals in the test population, with an age range of 1–17 years (M=29 and F=39). The results are presented in the table below:

All healthy Individuals

Instrument Mean (µg/L) 95th Percentile (µg/L) Number of Individuals

Phadia 100 3.8 8.2 163 Phadia 250 3.5 8.4 163

Adults Only

Instrument Mean (µg/L) 95th Percentile (µg/L) Number of Individuals

Phadia 100 4.7 9.9 95Phadia 250 4.4 8.9 95

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Pediatrics Only (<22 years old)

Instrument Mean (µg/L) 95th Percentile (µg/L) Number of Individuals

Phadia 100 2.8 6.4 68Phadia 250 2.4 6.3 68

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable

O. Conclusion:

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