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, or Phadia 1000.

Device Description

ImmunoCAP Tryptase is a fluorescence immunoassay for the measurement of human tryptase based on ImmunoCAP solid phase. ImmunoCAP Tryptase measures the total tryptase levels including all forma of a-tryptase and B-tryptase. ImmunoCAP Tryptase concentrations are quantitatively reported in microgram/L (ug/L).

ImmunoCAP Tryptase consists of assay specific reagents to be used as part of Phadia Laboratory Systems along with already cleared instruments (including instrument and data management software) and system reagents (system reagents were cleared in K051218).

ImmunoCAP Tryptase kits (for the Phadia 100, Phadia 250, and Phadia 1000) contain the following reagents:

ImmunoCAP Tryptase Conjugate
ImmunoCAP Tryptase Anti-Tryptase bound to ImmunoCAP carrier
Development solution
Stop solution
Washing solution
IgE/ECP/Tryptase sample diluent (available separately, not included in the ImmunoCAP Tryptase kit)
ImmunoCAP Tryptase Control (prepared from selected pooled human serum and lyophilized)

In addition, the Phadia 100 kit includes the following reagents:

Tryptase calibrators (1, 5, 12.5, 50, and 200 µg/L human tryptase in buffer)
Tryptase Curve Control 1 (single dose vials of human tryptase in buffer)

In addition, the Phadia 250 and Phadia 1000 kits include the following reagents:

ImmunoCAP Tryptase Calibrator Strip (1, 5, 12.5, 50, and 200 µg/L human tryptase in buffer)
Tryptase Curve Control Strip (human tryptase in buffer)

AI/ML Overview

The IMMUNOCAP TRYPTASE device is a fluorescence immunoassay for the measurement of human tryptase, intended as an aid in the clinical diagnosis of patients with a suspicion of systemic mastocytosis. The device's performance was evaluated through various studies, including precision, linearity, recovery, stability, detection limit, hook effect, analytical specificity, and clinical studies for sensitivity and specificity.

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria for precision were defined as a maximum total CVtotal% that would be met for all samples. The exact numerical criteria are obscured in the provided document (b)(4).

Study Type	Acceptance Criteria	Reported Device Performance
Precision	All samples should meet the specified CVtotal% (exact values obscured: (b)(4)).	Phadia 100: All acceptance criteria were met for 10 serum samples. Phadia 250: Criteria met for 14 out of 16 samples. Two very low-range samples were marginally out of specification but considered acceptable due to values being below clinical decision points. Phadia 1000: All specifications for CVtotal% were met for 15 serum samples.
Lot-to-lot Reproducibility	Not explicitly stated but implied to be within acceptable variability for reproducibility.	Demonstrated consistent results across different combinations of ImmunoCAP Tryptase Conjugate and Anti-Tryptase lots. The reported "Total%CV" values for 7 samples are obscured.
Linearity/Reportable Range	Recoveries should be within the range (0)(4).	Recoveries were within the range (0)(4) except for some intermediate dilutions of one sample (134 ug/L).
Recovery	Recovery values should meet the pre-determined specification of ®(%).	Recovery values met the pre-determined specification of ®(%).
Detection Limit (LoD)	LoD should be ®(4).	The LoB and LoD for each of the three instruments (Phadia 100, 250, 1000) were reported as being met (values obscured).
Hook Effect	No hook effect should be detected for samples with tryptase concentrations approximately 10 times the highest calibrator.	No hook effect was detected for any of the five tested samples (two serum, one EDTA plasma, one lithium heparin plasma, one sodium heparin plasma).
Analytical Specificity	All samples should exhibit recoveries of (b) (4) for interferences and (4) for spiked/unspiked HAMA samples.	All samples exhibited recoveries of (b) (4) for Bilirubin F & C, Chyle, Hemoglobin, and Rheumatoid Factor. No effect of heparin was observed. For HAMA, results met acceptance criteria (4) with one borderline exception.
Matrix Comparison	Plasma/serum ratios should fall within the range (4).	Serum vs. EDTA Plasma: Ratios for EDTA plasma/serum ranged (b)(4), consistent with acceptance criteria of (4). Serum vs. Lithium/Sodium Heparin Plasma: Ratios ranged (b)(4), consistent with acceptance criteria of (b)(4). plasma/serum ratio was within the range ®(4) for samples close to the assay cutoff.
Instrument Comparison	Acceptance criteria for the natural log of the ratio of mean tryptase results between instruments were (b)(4).	All results for ln(250/100), ln(1000/100), and ln(250/1000) (values obscured) were consistent with the acceptance criteria.
Clinical Sensitivity (Adult)	Not explicitly stated as a numerical acceptance criterion, but evaluated using a 20 ug/L cutoff.	Without Tryptase Consideration: 80.9% (95% CI: 71.5% to 87.7%) With Tryptase Consideration: 81.5% (95% CI: 72.4% to 88.1%)
Clinical Specificity (Adult)	Not explicitly stated as a numerical acceptance criterion, but evaluated using a 20 ug/L cutoff.	Without Tryptase Consideration: 44.9% (95% CI: 31.8% to 58.7%) With Tryptase Consideration: 47.8% (95% CI: 34.1% to 61.9%)
Clinical Sensitivity (Pediatric)	Not explicitly stated as a numerical acceptance criterion, but evaluated using a 20 ug/L cutoff.	Without Tryptase Consideration: 82.6% (95% CI: 62.9% to 93.0%) With Tryptase Consideration: 85.2% (95% CI: 67.5% to 94.1%)
Clinical Specificity (Pediatric)	Not explicitly stated as a numerical acceptance criterion, but evaluated using a 20 ug/L cutoff.	Without Tryptase Consideration: 97.0% (95% CI: 92.5% to 98.8%) With Tryptase Consideration: 100.0% (95% CI: 97.1% to 100.0%)

2. Sample Size Used for the Test Set and Data Provenance

Precision (Phadia 100): 10 serum samples.
Precision (Phadia 250): 16 serum samples.
Precision (Phadia 1000): 15 serum samples.
Linearity: 6 serum samples.
Hook Effect: 5 samples (two serum, one EDTA plasma, one lithium heparin plasma, one sodium heparin plasma).
Analytical Specificity: Two studies each with two serum samples (13.2 µg/L and 1.8 µg/L tryptase) and three serum samples (with tryptase levels (b)(4)). Heparin interference tested with blank and tryptase-spiked samples. HAMA tested with serum samples.
Matrix Comparison:
- Serum and EDTA Plasma: 21 healthy patients.
- Serum, Lithium Heparin Plasma, and Sodium Heparin Plasma: 20 healthy patients.
Instrument Comparison: 50 individual samples, 3 pooled internal control samples, and two lots of ImmunoCAP Tryptase Control.
Clinical Studies (Adults): 138 adult patients. Data provenance: Samples collected at one site in Spain over a 3-year period (retrospective/prospective not explicitly stated, but "over a 3-year period" suggests collection over time).
Clinical Studies (Pediatric): 156 pediatric patients. Data provenance: Samples collected at one site in Spain over an eight-year period (2003-2011).
Expected Values/Reference Range: 124 healthy individuals (89 adults, rest children).

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The ground truth for the clinical studies (systemic mastocytosis diagnosis) was established based on the WHO recommendations for mastocytosis, primarily the 4th edition of "WHO classification of tumors of hematopoietic and lymphoid tissues from 2008".

The studies mention the "Spanish Network on Mastocytosis" coordinated sample collection and patient classification. The WHO criteria themselves are based on a consensus proposal from "a large number of patients with established mastocytosis in different centers of Europe and North America" and involved a "consensus meeting 'Year 2000 Working Conference on Mastocvtosis'".

It is not specified how many individual experts established the ground truth for the specific 138 adult and 156 pediatric cases used in these studies, nor their individual qualifications (e.g., specific years of experience for radiologists). However, the ground truth is anchored in an internationally recognized consensus process and WHO classification, indicating a high level of expert involvement in developing the criteria.

4. Adjudication Method for the Test Set

The diagnostic classification for systemic mastocytosis was done "according to the WHO recommendations for mastocytosis, without consideration of the fourth minor criterion of tryptase levels persistently exceeding 20 ug/L" for the initial analysis. A reanalysis then included tryptase results to see the impact on classification.

This indicates an adjudication method based on an established medical guideline (WHO criteria), where the device's output (tryptase level) was initially excluded from the diagnosis to evaluate its standalone performance against the other criteria, and then included to reflect its intended use. There is no mention of a traditional reader adjudication method like 2+1 or 3+1 involving human readers explicitly reviewing cases to establish ground truth for each test case. Instead, the ground truth is derived from a set of clinical and laboratory findings interpreted by clinicians/pathologists following WHO guidelines.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. The studies evaluate the analytical performance of the automated ImmunoCAP Tryptase assay and its clinical effectiveness (sensitivity and specificity) in diagnosing systemic mastocytosis based on a WHO-defined cutoff, not the improvement of human readers with AI assistance. This device is an in vitro diagnostic test, not an AI-powered image analysis tool that assists human readers.

6. If a Standalone Study Was Done

Yes, standalone (algorithm only) performance was done. The precision, linearity, recovery, detection limit, hook effect, analytical specificity, and matrix/instrument comparison studies all assess the analytical performance of the ImmunoCAP Tryptase assay system itself, independent of human interpretation or assistance.

The clinical studies also present the device's performance (sensitivity and specificity) using a predefined cut-off (20 ug/L) against a clinical diagnosis established by other WHO criteria, which can be considered a standalone evaluation of its diagnostic utility.

7. The Type of Ground Truth Used

The ground truth used for the clinical studies was expert consensus / clinical diagnosis based on established guidelines. Specifically, the diagnosis of Systemic Mastocytosis was classified "according to the WHO recommendations for mastocytosis." These recommendations are themselves based on a consensus proposal developed from extensive clinical and laboratory data, and reviewed scientific literature. The criteria include major criteria (e.g., mast cell infiltrates in organs) and minor criteria (e.g., abnormal mast cell morphology, C-kit mutation, CD2/CD25 expression), excluding tryptase levels for the initial evaluation of the device.

8. The Sample Size for the Training Set

The document does not explicitly state a sample size for a training set. This is a diagnostic assay, and its performance characteristics (precision, linearity, etc.) are typically established through analytical studies using characterized samples rather than a "training set" in the machine learning sense. The clinical studies used patient samples for validation.

The "Traceability" section mentions that "The original reference standard was produced in-house and its concentration determined by the supplier. Each subsequent lot is standardized against reference standards." This implies an internal process for calibrating and standardizing the assay, which might involve a form of internal "training" or calibration, but not a distinct "training set" of patient data for algorithm development as seen in AI/ML products.

9. How the Ground Truth for the Training Set Was Established

As there is no explicitly defined "training set" for an algorithm in the context of an FDA submission for a fluorescence immunoassay, the concept of establishing ground truth for a training set is not directly applicable here. The assay relies on a biochemical reaction and a predefined calibration curve. The reference standards mentioned in the traceability section are used to standardize the assay, and their concentrations are determined by the supplier, implying analytical validation rather than expert-labeled ground truth in a clinical context.

Summary

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION

DECISION SUMMARY

A. 510(k) Number:

K103039

B. Purpose for Submission:

New analyte

C. Measurand:

Tryptase

D. Type of Test:

Fluorescence immunoassay

E. Applicant:

Phadia US Inc

F. Proprietary and Established Names:

ImmunoCAP Tryptase

G. Regulatory Information:

1. Regulation section:
  21 CFR §866.5760 – Tryptase test system
1. Classification:
  Class II
1. Product code:
  OYL, tryptase assay system
1. Panel:
  Immunology (82)

H. Intended Use:

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1. Intended use(s):

ImmunoCAP Tryptase is to be used with the instruments Phadia 100, Phadia 250, or Phadia 1000.

1. Indication(s) for use:
  Same as intended use.
1. Special conditions for use statement(s):
  Prescription use only.
1. Special instrument requirements:
  For use with the Phadia 100, Phadia 250, or Phadia 1000 instrument.

I. Device Description:

ImmunoCAP Tryptase kits (for the Phadia 100, Phadia 250, and Phadia 1000) contain the following reagents:

ImmunoCAP Tryptase Conjugate (4) (4) ● ImmunoCAP Tryptase Anti-Tryptase (b) (4) . bound to ImmunoCAP carrier Development solution ""(4) .
Stop solution (b) (4) ●
Washing solution ●
IgE/ECP/Tryptase sample diluent (available separately, not included in the ImmunoCAP ● Tryptase kit)
ImmunoCAP Tryptase Control (prepared from selected pooled human serum and

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lyophilized)

In addition, the Phadia 100 kit includes the following reagents:

Tryptase calibrators (1, 5, 12.5, 50, and 200 µg/L human tryptase in buffer) ●
Tryptase Curve Control 1 (single dose vials of human tryptase in buffer)

In addition, the Phadia 250 and Phadia 1000 kits include the following reagents:

. ImmunoCAP Tryptase Calibrator Strip (1, 5, 12.5, 50, and 200 µg/L human tryptase in buffer)
. Tryptase Curve Control Strip (human tryptase in buffer)

J. Substantial Equivalence Information:

1. Predicate device name(s):
  None
1. Predicate 510(k) number(s):
  None
1. Comparison with predicate:
  Not applicable.

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

Not applicable

L. Test Principle:

Anti-tryptase, covalently coupled to ImmunoCAP, reacts with the 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 for the patient samples are transformed to concentrations with the use of a calibration curve.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:
- a. Precision/Reproducibility:

Precision studies were conducted in alignment with CLSI EP5-A2. Precision studies were conducted on the Phadia 100, Phadia 250, and Phadia 1000 instruments. Total

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imprecision was calculated as the square root of the sum of the squares of each imprecision component (between instruments, between runs, within run).

The acceptance criteria for precision were:

Image /page/3/Figure/2 description: The image shows text excerpts from a document, likely related to medical or scientific data. The text includes labels such as "Phadia 100" and "Phadia 250 and Phadia 1000", suggesting different categories or groups being analyzed. The term "CVtotal%" is repeated multiple times, indicating it is a key metric or variable being measured and reported for each category. There is also a reference to "(b) (4)" which may be a footnote or citation.

Phadia 100: Ten serum samples with different levels of tryptase were assayed on three different instruments. Each sample was tested in multiple runs (9 runs for samples #1-5, 6 runs for samples #6-10). Four replicates were tested per run. All acceptance criteria were met. The results are shown in the following table:

Sample	N	Meanµg/L	BetweenInstruments%CV	BetweenRuns%CV	WithinRun%CV	Total%CV
1	108	(b) (4)
2	108
3	108
4	108
5	108
6	72
7	72
8	72
9	72
10	72
Sample	Meanµg/L	BetweenInstruments%CV	BetweenRuns%CV	WithinRun%CV	Total%CV
1	2	(b) (4)
2	2
3	4
4	8
5	18
6	34
7	59
8	99
9	110
10	114
11	178
12	21
13	20
14	19
15	182
16	173
Sample	N	Meanμg/L	BetweenInstruments%CV	BetweenRuns%CV	WithinRun%CV	Total%CV
1	72	(b) (4)
2	69*
3	72
4	72
5	72
6	72
7	72
8	72
9	72
10	72
11	72
12	72
13	72
14	72
15	72

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Phadia 250: The precision results are presented in the following table. 16 different serum samples with different levels of tryptase were assayed on three different instruments. Each sample was tested in 6 runs, and four replicates were tested per run. Acceptance criteria for CVtotal% were met for all samples, with the exception of two samples in the very low range (Sample 1 and 2), which were both marginally out of specification. The result was considered acceptable since tryptase values in these samples are well below any relevant clinical decision points.

Phadia 1000: The precision results for Phadia 1000 are presented in the following table. 15 different serum samples with different levels of tryptase were assayed on two different instruments. Each sample was tested in 9 runs, and four replicates were tested per run. All specifications for CV total% were met for all samples. Since only two

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instruments were used to evaluate the first 10 samples, CV between instruments is not applicable for those samples.

*three observations less due to laboratory error

Lot-to-lot reproducibility: Seven different samples with tryptase concentrations ranging 8-192 µg/L were tested using 3 different lots of ImmunCAP Tryptase Conjugate and 3 different lots of ImmunoCAP Tryptase Anti-Tryptase in different combinations of conjugate and anti-tryptase. All assays were performed on the Phadia 250 instrument. Each sample was tested with each of five different reagent combinations in 3 assay runs, with 3 replicates per run. The data are summarized in Table 4:

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Sample	Meanμg/L	BetweenLots %CV	Between Runs%CV	Within Run%CV	Total%CV
1	(b) (4)
2
3
4
5
6
7

b. Linearity/assay reportable range:
Linearity experiments were performed according to CLSI EP6-A. Six different serum samples with tryptase levels ranging 30 µg/L to 194 µg/L were manually diluted in two-fold dilutions in near tryptase free serum (tryptase level of 0.46 µg/L). The samples were tested with ImmunoCAP Tryptase in 2 replicates in 1 assay run.

The assays were performed according to ImmunoCAP Tryptase Directions for Use using the Phadia 250 instrument. Recoveries were in the range (0)(4) except for some of the intermediate dilutions for the 134 ug/L sample.

Sample	Dilution	ObservedMean (µg/L)	ExpectedMean (µg/L)	Recovery(%)
	1:1	(b) (4)
	1:2
	1:4
	1:8
194 µg/L	1:16
	1:32
	1:64
	1:128
Sample	Dilution	ObservedMean (µg/L)(b) (4)	ExpectedMean (µg/L)	Recovery(%)
169 µg/L	1:1
169 µg/L	1:2
169 µg/L	1:4
169 µg/L	1:8
169 µg/L	1:16
169 µg/L	1:32
169 µg/L	1:64
169 µg/L	1:128
134 µg/L	1:1
134 µg/L	1:2
134 µg/L	1:4
134 µg/L	1:8
134 µg/L	1:16
134 µg/L	1:32
134 µg/L	1:64
101 µg/L	1:1
101 µg/L	1:2
101 µg/L	1:4
101 µg/L	1:8
101 µg/L	1:16
101 µg/L	1:32
73 µg/L	1:1
73 µg/L	1:2
Sample	Dilution	ObservedMean (µg/L)	ExpectedMean (µg/L)	Recovery(%)
	1:4
	1:8
	1:16
	1:32
30 µg/L	1:1
	1:2
	1:4
	1:8

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c. Recovery

A recovery study was performed on the Phadia 100 instrument using purified tryptase (with known concentration) spiked into serum samples containing different basal levels of tryptase. Recovery values are shown in the chart below. Recovery values
met the pre-determined specification of®(%

Sample	Level	Tryptase(µg/L), Serum+ Tryptase(A)	Tryptase(µg/L) Serumonly(B)	Tryptase(µg/L) Buffer+ Tryptase(C)	Recovery(%)A/(B+C)
1	1
	2
	3
2	1
	2
	3
3	1
	2
	3

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

Traceability: There is no internationally traceable standard for tryptase. The original reference standard was produced in-house and its concentration determined by the supplier. Each subsequent lot is standardized against reference standards. New reference standard is produced approximately every three years.

Stability: Real-time stability was evaluated for ImmunoCAP Tryptase Anti-Tryptase and for the ImmunoCAP Tryptase Conjugate. Three independent preparations of Anti-Tryptase were stored at 2-8°C and were tested at various time points using the Phadia 100 instrument. Anti-tryptase was shown to be stable for up to 25 months when stored at this temperature.

Three independent preparations of ImmunoCAP Tryptase Conjugate were stored at 2-8°C and were tested at various time points using the Phadia 100 instrument. Conjugate was shown to be stable for up to 25 months when stored at this temperature.

On board stability of open ImmunoCAP Tryptase Conjugate bottles on the Phadia 250 instrument was determined by storing the conjugate under transport-simulated conditions. Conjugate was stored at 32°C for one week, followed by movement to 2-8°C for two 18-24 hour periods, then stored at 2-8°C. Prior to each test occasion (0, 13, and 25 months), the conjugate bottles were uncapped for 7 days at 2-8℃ to simulate on-board conditions. The results demonstrated that ImmunoCAP Tryptase Conjugate was stable under these conditions.

The short term stability of samples was investigated. Samples from 15 self-reported healthy volunteers containing various levels of tryptase were used for this study. The assays were performed using the Phadia 250 instrument. The results of the study demonstrated that samples could be stored at room temperature for up to 48 hours, could be frozen and thawed for up to 5 cycles, and could be stored at 2-8°C for up to 7 days.

ImmunoCAP Tryptase Calibrators and Calibrator Strip:

The raw material for ImmunoCAP Tryptase Calibrators and Curve Control is tryptase protein purified from human lung. For the Phadia 100, calibrators at five levels (1: 5; 12.5; 50 and 200 µg/L) are provided in individual vials. For the Phadia 250 and Phadia 1000, the ImmunoCAP Tryptase Calibrator Strip contains calibrators at five levels (1: 5: 12.5: 50 and 200 ug/L).

The stability of the ImmunoCAP Tryptase Calibrators (as used on the Phadia 100) was investigated. Three lots of calibrators were tested. The assays were performed using the Phadia 100 instrument. Each lot was stored under transport-simulated conditions-at 30°C for one week followed by 25 months at 2-8°C. The calibrators were found to be stable for up to 25 months when stored under these conditions.

An on-board stability study was performed with the ImmunoCAP Tryptase Calibrator

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Strip. This study is applicable to the Phadia 250 and Phadia 1000 instruments. One lot of ImmunoCAP Tryptase Calibrator Strip was transport-simulated by storage at 32℃ for one week, followed by movement to 2-8℃ for two 18-24 hour periods, then stored at 2-8℃. To demonstrate on-board stability, the calibrator strip was stored at 32°C for 28 days. The studies demonstrated that calibrators in the strip were stable under these conditions.

ImmunoCAP Tryptase Curve Control and Curve Control Strip:

The ImmunoCAP Tryptase Curve Control is packaged in either a vial format (Phadia 100) or the strip format (Phadia 250 and Phadia 1000) which consists of the same material as two of the five calibrators. The Curve Controls are automatically performed with each new run as a check on the overall curve performance. The stability data for calibrators also applies to this material.

ImmunoCAP Tryptase Control:

The stand-alone ImmunoCAP Tryptase Control is prepared using tryptase purified from human lung. Three lots of (6) 4) were tested utilizing the Phadia 100 instrument. The results showed that 10(4) ImmunoCAP Tryptase Control is stable for 26 months when stored at 2-8°C, and (0)(4) control is stable for 4 weeks when stored at 2-8℃.

e. Detection limit:
Limit of detection (LoD) studies were performed as follows. 24 replicates of the ImmunoCAP IgE/ECP/Trptase Sample Diluent and 24 replicates of the lowest ImmunoCAP Tryptase Calibrator (1 ug/L) were tested using the Phadia 100, Phadia 250, and Phadia 1000 instruments in one assay run each. The acceptance criteria were that the LoD should be ®(4) Calculations of Limit of Blank (LoB) and LoD were performed according to CLSI EP17-A. The table below summarizes the results of the Limit of Detection studies. Calculations were performed on Fluorescence Response Units (RU) since no tryptase concentrations are calculated below the lowest calibrator.

Instrument	MeanCalibrator 1(RU)	Mean Samplediluent(RU)	LoB(RU)	LoD(RU)
Phadia 100	(b) (4)
Phadia 250
Phadia 1000

The results showed that the LoB and LoD for each of the three instruments were

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f. Hook effect:
Five samples from different matrices (two serum samples and one sample each of EDTA plasma, lithium heparin plasma, and sodium heparin plasma) with tryptase concentrations approximately 10 times the highest ImmunoCAP Tryptase calibrator point (b) (4) were used to assess any potential hook effects. No hook effect was detected for any of these samples.
g. Analytical specificity:
In one study, interference testing performed with two serum samples, 13.2 µg/L and 1.8 µg/L tryptase with Bilirubin F (b)(4) Bilirubin C (b) (4) Chyle (b) (4) , Hemoglobin (b) (4) and Rheumatoid

Factor (6) (4) All samples exhibited recoveries of (b) (4)

In a second study, interference testing performed with three serum samples, 6) (4) of tryptase with Bilirubin F (4) ). Bilirubin C ,(b) (4) Chyle (b) (4) Hemoglobin (6) (4) and ). All samples exhibited recoveries of (6) (4) Rheumatoid Factor (b) (4)

Interference testing was also performed with heparin (concentrations ranging ®(4) ) spiked into blank samples, as well as in samples containing tryptase levels ranging 6) (4) All samples exhibited recoveries of (4) . No effect of heparin was observed on blank samples.

Interference testing was also performed with human anti-mouse antibodies (HAMA) with concentrations up to (0) (4) spiked into serum samples containing tryptase levels ranging from (b) (4) Two different studies were performed, each using a different master lot of ImmunoCAP Tryptase assay reagents and using the Phadia 250 system. The results from the studies met the acceptance criteria (4) observed/expected for spiked sample/unspiked sample), with the exception of one borderline result (b)(4) for a sample containing (6) (4

h. Assay cut-off:
The cut-off limit of 20 ug/L tryptase is internationally accepted as a minor criterion for systemic mastocytosis, and is defined by the World Health Organization Classification of Tumors. The WHO classification criteria are based on a consensus process as described in the introduction of the present 4th edition of "WHO classification of tumors of hematopoietic and lymphoid tissues from 2008'. The present valid criteria are described in a separate chapter in this volume".

4 Harris NL, Campo E, Jaffe S et.al. Introduction. In: 4th edition of tumours of haematopoietic and lymphoid tissues .: (eds) Swerdlow SH, Campo E, Harris NL et al., Lyon: IARC; 2008:14-15

2 Homy HP, Metcalfe DD, Bennet JM et al. Mastocytosis. In: 44 edition of WHO classification of tumours of haematopoietic and lymphoid tissues .: (eds) Swerdlow SH, Campo E, Harris NL et al., Lyon: IARC, 2008: 54-63.

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The consensus proposal, citing 175 scientific publications spanning over several decades, was published in 2001.

The basis of the consensus proposal, i.e. patients, parameters, and analyses are described in Valent et al.3: The consensus proposal is a retrospective analysis of a large number of patients with established mastocytosis in different centers of Europe and North America. In addition, a larger number of control cases without mastocytosis (myeloid neoplasms) were examined. In all cases, clinical findings, laboratory findings, histologic and immunohistologic data were collected for all patients. The clinical course was compared to, and correlated with, laboratory and histologic parameters. Data from almost all patients have been published previously. In addition, the entire literature was reviewed whenever possible to compare and discuss their findings.

The proposal was discussed and accepted by WHO at a final consensus meeting "Year 2000 Working Conference on Mastocvtosis" and the WHO criteria were published 2001 in the 3td edition of "WHO classification of tumors of hematopoietic and lymphoid tissues'4 and were then transferred into the 4th edition', published in 2008.

The WHO consensus Diagnostic Criteria for Systemic Mastocytosis (SM) are listed as follows:

If at least 1 major and 1 minor, or at least 3 minor criteria, are met, the diagnosis of Systemic Mastocytosis (SM) can be established.

Major Criteria: Multifocal dense infiltrates of mast cells in bone marrow or other extracutaneous organ(s) (>15 mast cells in aggregate).

Minor Criteria:

a) Mast cells in bone marrow or other extracutaneous organ(s) show an abnormal morphology (> 25%)
b) C-kit mutation at codon 816 in extracutaneous organ(s). (Activating mutations at codon 816; in most cases, c-kit D816V)
c) Mast cells in bone marrow express CD2 and/or CD25
d) Serum total tryptase > 20 µg/L (does not count in patients who have associated hematologic clonal non-mast cell lineage disease-type disease).

3 Valent P., Horny HP., Escribano L., et al. Diagnostic criteria and classification of mastocytosis: a concensus proposal. Leukemia Research 2001; 25: 603-625.

4 Valent P, Horny H, Li C, et al: Mastocytosis. In: 3rd edition of World health organization of tumors. Pathology and genetics of tumors of hematopoietic and lymphoid tissue. (eds) Jaffe E. Harris N, Stein H, Vardiman J, Lyon, LARC Press, 2001: 293-302. (Not available, replaced by 4th edition)

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2. Comparison studies:

a. Method comparison with predicate device:
Not applicable.
b. Matrix comparison:
Two matrix comparison studies were performed on the Phadia 250 instrument. All samples were tested with two replicates in one assay run.

The first matrix comparison study (serum and EDTA plasma) was performed using samples from 21 healthy patients with tryptase levels ranging from 60(4) Ratios for EDTA plasma/serum ranged あな , consistent with acceptance criteria of (4) for all samples.

The second matrix comparison study (serum, lithium heparin plasma and sodium heparin plasma) was performed using samples from 20 healthy patients with tryptase levels ranging from (b) (4) . Ratios for lithium heparin plasma/serum and sodium heparin plasma/serum ranged (6) (4) . consistent with acceptance criteria of (b) (4) for all samples.

For both studies, the plasma/serum ratio was within the range ®(4) for samples close to the assay cutoff of 20 ug/L. Deming regression was performed for each comparison. Slopes and intercents from this regression analysis are shown in the table below.

Deming Regression Analysis		Estimate
Serum vs	Intercept	(b) (4)
EDTA plasma	Slope
Serum vs	Intercept
lithium heparin plasma	Slope
Serum vs	Intercept
sodium heparin plasma	Slope

c. Instrument comparison:

To determine the conformity between the Phadia 100, Phadia 250, and Phadia 1000 instruments, the following study was conducted. The ImmunoCAP Tryptase Calibrators, 50 individual samples, 3 pooled internal control samples and two lots of ImmunoCAP Tryptase Control were assayed in 6 replicates with four different

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Phadia 100 instruments (3 runs each). 3 different Phadia 250 instruments (2 runs each) and 3 different Phadia 1000 instruments (2 runs each). For each sample, the natural log of the ratio of the mean tryptase results between the two instruments was calculated (for example, to compare the Phadia 100 and Phadia 250 results, the calculation was In(mean result250/mean result100). Acceptance criteria were

for this calculation. Samples that tested above the reportable range (0(4) were not included in the calculations. The results are summarized as follows:

Conformity calculation	Average result
$ln(250/100)$	(b) (4)
$ln(1000/100)$
$ln(250/1000)$

A Deming regression analysis of the instrument conformity data was also performed, with the following results:

Phadia InstrumentComparison	Intercept (95%CI)(ug/L tryptase)	Slope (95% CI)
Phadia 100 vs. Phadia 250	(b) (4)
Phadia 1000 vs. Phadia 250

3. Clinical studies:

a. Clinical Sensitivity and Specificity:

Adult patients

Samples from 138 adult patients (63 male and 75 female, age range 24-81) with a suspicion of mastocytosis (all comers) were collected at routine patient visits over a 3-year period. The reasons for referral included skin lesions (61 patients), anaphylaxis (54 patients), mast cell activation (15 patients), and other clinical findings (8 patients). ImmunoCAP Tryptase assays were performed on these samples, using the Phadia 250 instrument. Sample collection was coordinated by the Spanish Network on Mastocytosis and performed at one site in Spain. Patients were classified according to the WHO recommendations for mastocytosis, without consideration of the fourth minor criterion of tryptase levels persistently exceeding 20 ug/L.

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		Systemic Mastocytosis(without tryptase consideration)
		+		Total
ImmunoCAPTryptase Assay(20 ug/L cutoff)	+	72	27	99
		17	22	39
	Total	89	49	138

Sensitivity (72/89) = 80.9% (95% CI: 71.5% to 87.7%) Specificity (22/49) = 44.9% (95% CI: 31.8% to 58.7%)

Elevated tryptase level is one of several minor criteria for a diagnosis of mastocytosis. Tryptase levels are known to be increased in a number of disorders apart from mastocytosis, e.g. in AML patients and in patients with CML and other myeloid neoplasms not classified as systemic mastocytosis. This results in a low (~45%) specificity of the tryptase assay in adults if used as the sole criterion for mastocytosis. However, since tryptase is used in conjunction with other clinical and diagnostic criteria, tryptase is a useful diagnostic tool despite of its low specificity in adults.

The data from the adult clinical study was reanalyzed to include the tryptase result in the calculations. Inclusion of tryptase would have changed the diagnosis for three patients:

		Systemic Mastocytosis(with tryptase consideration)
		+	-	Total
ImmunoCAPTryptase Assay(20 µg/L cutoff)	+	75	24	99
	-	17	22	39
	Total	92	46	138

Sensitivity (75/92) = 81.5% (95% CI: 72.4% to 88.1%) Specificity (22/46) = 47.8% (95% CI: 34.1% to 61.9%)

Pediatric patients:

Samples from 156 pediatric patients (ages <1-19) with a suspicion of mastocytosis were collected at routine patient visits over an eight year period (2003-2011). The type of symptoms leading to referral included maculopapular (76 patients), nodular

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(34 patients), diffuse (19 patients), mastocytoma (18 patients), and plaque (9 patients). ImmunoCAP Tryptase assays were performed on these samples, using the Phadia 250 instrument. Sample collection was coordinated by the Spanish Network on Mastocytosis and performed at one site in Spain. Patients were classified according to the WHO recommendations for mastocytosis, without consideration of the fourth minor criterion of tryptase levels persistently exceeding 20 ug/L.

		Systemic Mastocytosis(without tryptase consideration)
		+	-	Total
ImmunoCAPTryptase Assay(20 ug/L cutoff)	+	19	4	23
	-	4	129	133
	Total	23	133	156

Sensitivity (19/23) = 82.6% (95% CI: 62.9% to 93.0%)

Specificity (129/133) = 97.0% (95% CI: 92.5% to 98.8%)

The data from the pediatric clinical study was reanalyzed to include the tryptase result in the calculations. Inclusion of tryptase would have changed the diagnosis for four patients:

		Systemic Mastocytosis(with tryptase consideration)
		+	-	Total
ImmunoCAPTryptase Assay(20 µg/L cutoff)	+	23	0	23
	-	4	129	133
	Total	23	133	156

Sensitivity (23/27) = 85.2% (95% CI: 67.5% to 94.1%)

Specificity (129/129) = 100.0% (95% CI: 97.1% to 100.0%)

Children seen by physicians for the suspicion of mastocytosis do not typically have other diseases that may elevate tryptase levels (e.g. AML, CML) to the same extent as adults. Therefore the clinical specificity for tryptase is higher in children than in adults.

The diagnosis of systemic mastocytosis has been established using worldwide criteria that included input from U.S. clinicians and scientists. The population demographics

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are similar between Spain and the U.S. Systemic mastocyosis occurs in all ethnic groups, but is seen more often in Caucasians .

b. Other clinical supportive data (when a. is not applicable):
Not applicable.
1. Clinical cut-off:
  See Assay Cut-off.
1. Expected values/Reference range:
  Serum samples from 124 healthy individuals between 3-67 years of age were analyzed in duplicate using the ImmunoCAP Tryptase assay on the Phadia 250 instrument. The results showed that tryptase concentrations in this population had a mean concentration of tryptase of 4.4 µg/L and an upper 95th percentile at 12.0 µg/L.

The results for adults only (89 individuals, ages 22-67) had a mean concentration of tryptase of 5.0 µg/L and an upper 95th percentile at 13.5 µg/L. Thus, there were no major differences observed in expected values between healthy adults and healthy children, consistent with data reported in the literature'.

N. Proposed Labeling:

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

O. Conclusion:

The petition for Evaluation of Automatic Class III Designation for this device is accepted. The device is classified as Class II under regulation 21 CFR 866.5760 with special controls. The special control guidance document "Class II Special Controls Guidance Document: Tryptase Test System as an Aid in the Diagnosis of Systemic Mastocytosis" will be available shortly.

S Komarow, HD et al. (2009) J. Allergy Clin. Immunol 124 (4), 845-848

Regulation Number and Section

§ 866.5760 Tryptase test system.

(a)
Identification. A tryptase test system is a device that aids in the diagnosis of systemic mastocytosis. 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.(b)
Classification. Class II (special controls). The special control is FDA's guideline entitled “Class II Special Controls Guideline: Tryptase Test System as an Aid in the Diagnosis of Systemic Mastocytosis.” For availability of the document, see § 866.1(e).