K Number

Device Name

PHICAL TEST

Manufacturer

GENOVA DIAGNOSTICS

Date Cleared

2006-04-26

(34 days)

Product Code

NXO CAL

Regulation Number

866.5180

Intended Use

The PhiCal™ test is a quantitative ELISA for measuring, in human stool, concentrations of fecal calprotectin, a neutrophilic protein that is a marker of mucosal inflammation. The PhiCal™ test can be used as an in vitro diagnostic to aid in the diagnosis of inflammatory bowel diseases (IBD): Crohn's disease and ulcerative colitis, and to differentiate IBD from irritable bowel syndrome; when used in conjunction with other diagnostic testing and the total clinical picture.

Device Description

The PhiCal test assay consists of: microtiter plate coated with polyclonal rabbit antibodies for calprotectin (12 strips, 8 wells per strip); alkaline phosphatase labeled rabbit anti-calprotectin IgG in buffer with Proclin 300; substrate in buffer; 20X washing solution; 10X dilution solution with Proclin 300; 5X extraction solution with Proclin 300; 5 vials of calprotectin solution at concentrations of 6.25, 12.5, 25, 50 and 100 ng/mL; a low and a high control.

More Information

Contact

Type

Center

Panel

Date Received

Product Code

Subsequent Product Codes

Applicant Name (Manufacturer)

Device Name

Decision

Street 1

Street 2

City

State

Country Code

ZIP

Postal Code

Class Advise Committee

SSP Indicator

Third Party Reviewed

Expedited Review

Predicate Devices

Not applicable

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies describe a standard ELISA assay for measuring a biomarker in stool. There is no mention of AI or ML in the text.

Therapeutic

No
The device is an in vitro diagnostic test used to measure markers for disease, not directly treat or prevent a disease.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that "The PhiCal™ test can be used as an in vitro diagnostic to aid in the diagnosis of inflammatory bowel diseases (IBD)". This indicates its role in identifying or differentiating medical conditions, which is the primary function of a diagnostic device.

SaMD (Software as a Medical Device)

The device description clearly outlines a physical kit containing reagents, plates, and solutions for an ELISA test, indicating it is a hardware-based in vitro diagnostic device, not software only.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the PhiCal™ test "can be used as an in vitro diagnostic".
Nature of the Test: The test measures a substance (fecal calprotectin) in a biological sample (human stool) to aid in the diagnosis of diseases (IBD). This is the core function of an in vitro diagnostic device.
Device Description: The description details the components of an assay kit designed for laboratory testing of biological samples.
Clinical Studies: The inclusion of clinical studies evaluating sensitivity and specificity further supports its classification as a diagnostic device used in a clinical setting.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

Product codes (comma separated list FDA assigned to the subject device)

NXO

Device Description

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Stool / Fecal

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Precision/Reproducibility:

Intra-Assay precision: One low and one high matrix sample, each assayed twenty times within a single run. CV's were 4.07% and 6.17%. 12 samples assayed in 6 replicates, range of %CV was 2.9% to 14.3%.
Inter-assay precision: Six different pools of stool, 20 separate extraction and assay runs. %CV range was 8.9% to 18.1%. Ten different samples (5 positive, 5 negative), each extracted 5 separate times, 4 replicates on 5 separate EIA runs. %CV range was 5.8% to 20.1%.
Extraction Repeat Reproducibility: Two samples representing low and high ends of reportable range, each extracted 24 times. CV's were 12.60% and 12.13%.

Split Sample Comparison Study:

40 stool samples assayed by GENOVA and Fagerhol laboratory (Norway).
Comparison plot: y = 0.9603x + 9.7691, r- = 0.9618.

Linearity/Assay Reportable Range:

Aqueous linearity: 6.25 - 100 ng/ml. Equation: y = 0.8771x + 2.8302, R2=0.9962.
Matrix linearity: 7.73 - 114.90 ng/ml. Equation: y = 1.0569x -0.1658, R2=0.9866.

Accuracy/Recovery:

Extracts from five different stool samples spiked with calprotectin.
% Recovery ranged from 99.1% to 118.6%.

Detection Limit (Functional Sensitivity):

Minimum Detection Limit (MDT): Determined by running 20 replicates of assay buffer. Result was below functional sensitivity.
Functional sensitivity: Established at

Regulation Number and Section

§ 866.5180 Fecal calprotectin immunological test system.

(a)
Identification. A fecal calprotectin immunological test system is anin vitro diagnostic device that consists of reagents used to quantitatively measure, by immunochemical techniques, fecal calprotectin in human stool specimens. The device is intended forin vitro diagnostic use as an aid in the diagnosis of inflammatory bowel diseases (IBD), specifically Crohn's disease and ulcerative colitis, and as an aid in differentiation of IBD from irritable bowel syndrome.(b)
Classification. Class II (special controls). The special control for these devices is FDA's guidance document entitled “Class II Special Controls Guidance Document: Fecal Calprotectin Immunological Test Systems.” For the availability of this guidance document, see § 866.1(e).

Summary

510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number: K050007
B. Purpose for Submission: New device.
C. Measurand: Fecal calprotectin
D. Type of Test: Quantitative, ELISA
E. Applicant: Genova Diagnostics
F. Proprietary and Established Names: PhiCal™ Test

G. Regulatory Information:

1. Regulation section: None
1. Classification: De novo
1. Product Code: NXO, Calprotectin, fecal
1. Panel: Immunology (82)

H. Intended Use:

1. Intended use(s):
- The PhiCal™ test is a quantitative ELISA for measuring, in human stool, concentrations of fecal calprotectin, a neutrophilic protein that is a marker of mucosal inflammation. The PhiCal™ test can be used as an in vitro diagnostic to aid in the diagnosis of inflammatory bowel diseases (IBD): Crohn's disease and ulcerative colitis, and to differentiate IBD from irritable bowel syndrome.
1. Indication(s) for use:

1. Special condition for use statement(s): The device is for prescription use only.
1. Special instrument requirements: ELISA reader (405 nm filter), digital scale (40-150 mg), vortex mixer, shaker, micro-centrifuge
I. Device Description:

J. Substantial Equivalence Information:

1. Predicate device name(s): Not applicable
1. Predicate K number(s): Not applicable
1. Comparison with predicate:

Similarities
Item	Device	Predicate

Differences
Item	Device	Predicate

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

None referenced

L. Test Principle:

The test is performed on random stool samples, collected without preservatives. The samples should be tested within 7 days, or frozen at -20° C until tested. An extract is prepared by combining approximately 0.1 gm of stool with 5 mL of extraction buffer and mixing for 30 minutes. Following centrifugation, 20uL of the supernatant is diluted 1:50 with dilution buffer (final dilution 1:2500).

The assay uses a polyclonal rabbit antibody against calprotectin as the capture antibody in an enzyme linked immunosorbent assay system. Calprotectin present in the diluted sample is bound by the antibody adsorbed onto the surface of the microtiter plate. The enzyme conjugated antibodies (rabbit IgG antibodies against calprotectin) bind to the captured antigen. A substrate (alkaline phosphatase) is added and subsequently the enzyme catalyses the conversion of the substrate to a colored product. The intensity of the color is proportional to the amount of conjugate bound, and thus to the amount of captured calprotectin. The concentration of calprotectin in the samples is interpreted from a standard curve using 5 calibrators.

M. Performance Characteristics (if/when applicable):

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

Intra-Assay precision was determined by extracting one low and one high matrix sample, and assaying each extract twenty times within a single assay run. Precision is calculated as the % CV obtained for each level. Respective CV's were 4.07% and 6.17%. Additionally 12 samples were

assayed in 6 replicates within a singly assay run. Range of %CV was
2.9% to 14.3%. The resultant %CVs demonstrates that the PhiCal Test is
precise in the reportable range.

Calprotectin Intra-Assay Precision ( g/g)
		1	2	3	4	5	6	7	8	9	10	11	12
Analysis	Mean	177.5	55.3	229.4	31.1	96.9	106.8	17.5	18.7	20.8	182.8	55.6	60.2
	Std
Dev	5.7	6.6	6.7	2.3	11.5	9.4	0.8	2.7	1.9	10.8	3.9	2.5
	%CV	3.2	12.0	2.9	7.4	11.9	8.8	4.7	14.3	9.2	5.9	7.0	4.1

Inter-assay precision was assessed by evaluating results for six different pools of stool. Points are from 20 separate extraction and assay runs. The %CV range was 8.9% to 18.1%. Ten different samples (5 positive, 5 negative) were each extracted 5 separate times from individual stool aliquots. Each extract was then assayed in 4 replicates on 5 separate EIA runs performed on 5 different days. The %CV range was 5.8% to 20.1%. These results demonstrate the assav is reproducible within acceptable limits along the reportable range of the assay.

Calprotectin Inter-Assay Precision ( g/g)
Analysis	Replicate	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10
	Mean	36.9	12.9	16.6	20.2	26.9	131.7	188.2	89.8	101.1	63.3
	Std Dev	2.1	2.1	2.5	0.9	2.0	10.2	36.9	10.4	13.3	12.7
	%CV	5.8	16.7	14.9	4.4	7.6	7.7	19.6	11.6	13.2	20.1

Extraction Repeat Reproducibility: Two samples that represent the low and high ends of the reportable range were tested. Each was extracted 24 times, and each extract was tested. Precision was evaluated according to the %CV obtained for each level. Respective CV's of 12.60% and 12.13% for the low and high levels demonstrate that results are reproducible along the reportable range.

Split Sample Comparison Study:

Forty stool samples that spanned the reportable range of the test were assayed both by GENOVA and by the Fagerhol laboratory in Norway. The forty samples were assayed in small batches over 5 days to introduce day-to-day variability and to minimize any systematic error that might occur in a single run. The comparison plot showed y = 0.9603x + 9.7691, r- = 0.9618.

ii. Linearity/assay reportable range:

To confirm the reportable range of the PhiCal™ assay, standard curves were generated for both aqueous and matrix linearity. The results with serially diluted aqueous calprotectin in triplicate showed acceptable

linearity from 6.25 - 100 ng/ml. The following equation was obtained: Standard curve Aqueous, y = 0.8771x + 2.8302, R2=0.9962.

Image /page/3/Figure/2 description: The image is a graph titled "Calprotectin Aqueous Linearity 2-26-03". The graph plots "Observed ng/ml" on the y-axis versus "Theoretical ng/ml" on the x-axis. A black line is plotted through the data points, and the equation of the line is "y = 0.8771x + 2.8302" with an R-squared value of 0.9962.

The results with serially diluted fecal calprotectin in triplicate showed acceptable linearity and accuracy from 7.73 - 114.90 ng/ml. The following equation was obtained: Standard curve Matrix, y = 1.0569x -0.1658, R2=0.9866.

Image /page/3/Figure/4 description: The image is a scatter plot titled "Matrix Linearity Calprotectin". The x-axis is labeled "Theoretical Value ng/ml", and the y-axis is labeled "Observed Value ng/ml". The plot shows a linear relationship between the theoretical and observed values, with the equation of the line being y = 1.0569x - 0.1658 and R^2 = 0.9866.

Accuracy/ Recovery

Extracts from five different stool samples were each spiked with calprotectin. The calprotectin used for the spike was obtained from established serum pools. The baseline extract for each sample was "spiked" with assay buffer to compensate for volume adjustments made to

Calprotectin Recovery Data
# 1a

2a

3a

4a

5a | | | | | | | | | | | |

| Baseline (ug/g) | 49.1 | 73.4 | 66.4 | 6.4 | 73.6 | | | | | | |
| Spike Value (ug/g) | 58.5 | 57.6 | 55.2 | રરું રે | 59.0 | | | | | | |
| Theoretical (Base + Spike) (ug/g) | 107.6 | 131.0 | 121.7 | 61.7 | 132.7 | | | | | | |
| Observed (Base + Spike) (ug/g) | 106.7 | 129.9 | 144.3 | 66.1 | 140.0 | | | | | | |
| % Recovery | 99.2 | 99.1 | 118.6 | 107.2 | 105.5 | | | | | | |

the calprotectin-spiked extracts. Each of the spiked extracts was then assayed per kit protocol. Data is shown in table below.

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

Calibrators:

The primary internal reference is calibrated by comparison to the "Gold Standard" preparation provided by Dr. Magne Fagerhol, Norway.

Analyte Stability:

Studies were conducted to determine the stability of calprotectin in neat stool and in stool extracts. To determine stability in neat stool, several temperature conditions were evaluated: stool collected fresh and subsequently stored at 2-8°C: stool collected fresh and then stored at -20°C; and stool collected fresh and subjected to variable temperatures. Stability in stool extract was evaluated by extracting fresh samples and then storing aliquots of the extract at -20°Cm where a different aliquot was thawed for each analysis over 11 days.

For the variable temperature stability study the collected samples were stored at 2-8°C for 24 hours, after which they were stored in an incubator at 37°C for approximately 21 hours. Once removed from the incubator they were left at room temperature for approximately 5½ hours then stored at 2-8°C for the remainder of the study. This study concluded that calprotectin levels stay relatively consistent (no trends observed) for up to 11 days after collection, under conditions that may be experienced during specimen transport. The studies support the specimen collection, transport and storage recommendations in the package insert.

iv. Detection limit (functional sensitivity):

Minimum Detection Limit:

The minimum detection limit (MDT) was determined by running 20 replicates of the assay buffer (treated as patient) on one assay plate. Two standard deviations were added to the mean OD reading, and this value was plugged into the calibration curve to arrive at the MDT. The result was below the functional sensitivity of the assay.

Functional sensitivity:

The functional sensitivity was estimated by determining a precision profile at the low concentration range and then selecting the concentration at

which a 20% CV is obtained. To establish the functional sensitivity for the assay, 3 concentrations of kit standards, 12.5 ng/mL, 6.25 ng/mL and 3.125 ng/mL (6.25 ng/mL standard diluted 1:2 with dilution buffer), were assayed in either triplicate or quadruplicate over 6 days. %CV for all three concentrations were 120 mcg/gm | 254 | 29 | 283 |
| 50-120 mcg/gm | 43 | 89 | 132 |

| 120 µg/g | Abnormal | Repeat as clinically indicated |

1. Expected values/Reference range:
  The expected value in the normal population is