K Number
K173212
Device Name
Instant-view-PLUS immunochemical Fecal Occult Blood Test
Date Cleared
2018-02-15

(136 days)

Product Code
Regulation Number
864.6550
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP Authorized
Intended Use
The Instant-view-plus™ Immunochemical Fecal Occult Blood Test is a qualitative immunoassay for detection of Fecal Occult Blood. It is intended for professional and over the counter use.
Device Description
This device is a Driven Flow™ chromatographic immunoassay consisting of a test strip housed in a plastic cassette.
More Information

No
The device description and performance studies describe a standard immunochemical assay, with no mention of AI, ML, or image processing.

No
This device is a diagnostic test (qualitative immunoassay) used to detect fecal occult blood, which aids in disease screening or diagnosis rather than providing direct therapy.

Yes

This device is an immunochemical fecal occult blood test used for the detection of fecal occult blood, which is a diagnostic indicator for various gastrointestinal conditions. It provides a qualitative assessment, classifying it as a diagnostic tool.

No

The device description explicitly states it is a "Driven Flow™ chromatographic immunoassay consisting of a test strip housed in a plastic cassette," which are physical hardware components, not software.

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

Here's why:

  • Intended Use: The intended use explicitly states it's for the "detection of Fecal Occult Blood" using a "qualitative immunoassay." This is a test performed on a biological sample (feces) to provide information about a medical condition (presence of occult blood).
  • Device Description: It describes a "chromatographic immunoassay consisting of a test strip housed in a plastic cassette." This is a typical format for many IVD tests.
  • Performance Studies: The document details numerous performance studies (Precision/Reproducibility, Prozone Effect, Stability, Analytical specificity, Cross-Reactivity, Interfering Substances, etc.) which are standard requirements for demonstrating the analytical and clinical performance of an IVD.
  • Method Comparison with Predicate Device: Comparing the device's performance to existing, cleared IVD devices (predicate devices) is a key part of the regulatory process for IVDs.
  • Key Metrics: The document provides metrics like Percent Agreement, which are used to evaluate the performance of diagnostic tests.

All of these elements strongly indicate that the Instant-view-plus™ Immunochemical Fecal Occult Blood Test is an In Vitro Diagnostic device.

N/A

Intended Use / Indications for Use

The Instant-view-plus™ Immunochemical Fecal Occult Blood Test is a qualitative immunoassay for detection of Fecal Occult Blood. It is intended for professional and over the counter use.

Product codes

KHE

Device Description

This device is a Driven Flow™ chromatographic immunoassay consisting of a test strip housed in a plastic cassette.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

professional and over the counter use.

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

Precision/Reproducibility:
Repeatability was evaluated using three lots of devices, in-house by two trained laboratory professionals. For repeatability Hb-free fecal samples were collected and spiked with human whole blood with known hemoglobin levels to achieve the following six fecal hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 50 ng/ml, 55 ng/ml, and 500 ng/ml. Fifty replicates were performed for each concentration level.
Reproducibility was conducted across three intended use sites using three device lots (same three lots at all sites), and three operators (one at each site) over a minimum of 5 days. For reproducibility Hb-free fecal samples were collected and spiked with human whole blood with known hemoglobin levels to achieve the following nine fecal hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 50 ng/ml, 60 ng/ml, 60 ng/ml, and 500 ng/ml. Fourteen replicates were performed for each sample and concentration level. Insure® FITTM FOBT Controls (K101831) Negative and Positive Controls were also tested daily to ensure the validity of the test results.

Prozone Effect Study:
The Prozone effect was evaluated using three device lots, by two operators testing Hbfree stool specimens spiked with human blood of known hemoglobin concentrations so as to obtain fecal test samples with different Hb concentrations: 1,000 ng/ml (1 ug/ml), 2,000 ng/ml (2 ug/ml), 3,000 ng/ml (3 ug/ml), 4,000 ng/ml (4 ug/ml), 5,000ng/mL (5 ug/ml), 50,000 ng/mL (50 ug/ml), and 500,000 ng/mL (500 ug/ml). Twenty aliquots of each sample concentration were mixed with extraction buffer in the specimen collection tubes and tested in a randomized order.

Analytical specificity:
Specificity to human hemoglobin variant: The ability of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to detect human hemoglobin variants was determined by testing hemoglobin-A (HbA). hemoglobin-S (HbS) and hemoglobin-C (HbC) by preparing spiked stool samples containing seven different hemoglobin concentrations: 0 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. (3) aliquots of each of the seven concentrations (7 HbA, 7 HbS and 7 HbC supernatants) of spiked stool test samples were mixed with detection buffer in Instant-view-plus™ Immunochemical Fecal Occult Blood Test collection bottles. Samples were tested in a randomized order.

Cross-Reactivity: Cross-reactivity of Instant-view-plus™ Immunochemical Fecal Occult Blood Test with animal hemoglobin was evaluated by testing prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. Five replicates of spiked hemoglobin-negative stool concentrations listed above were then spiked with the intended level of respective animal hemoglobin such as beef (2000 ug/ml), chicken (500 ug/ml), fish (100 µg/ml), horse (500 ug/ml), goat (500 µg/ml), rabbit (500 µg/ml), pig (500 µg/ml), horseradish peroxidase (20.000 ug/ml), and sheep (100 ug/ml). All samples were tested in a randomized order.

Interfering Substances: Susceptibility of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to interference from extracts (vegetable) was evaluated by testing (5) replicates of prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml, 48 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. The 5 replicates of the spiked hemoglobin-negative stool concentrations listed above were spiked with the intended level of respective vegetable extracts: broccoli extracts (100%), cantaloupe extract (100%), cauliflower extract (100%), parsnip extract (100%), red radish extract (100%), and turnip extract (100%). All samples were mixed with detection buffer in Instant-view-plus™ Immunochemical Fecal Occult Blood Test sample collection bottles and tested in a randomized order.
Interfering Supplements: Susceptibility of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to interference from iron and sodium L-ascorbate was evaluated by testing 3 replicates of prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml. 48 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml. The 3 replicates of spiked hemoglobin-negative stool concentrations were spiked with iron (0.5% (w/v), and sodium L-ascorbate (0.5% (w/v). All samples were mixed with detection buffer in Instant-view-plus™ Immunochemical Fecal Occult Blood Test sample collection bottles and tested in a randomized order.

Interference from Toilet Water: Susceptibility of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to interference from toilet water was evaluated by testing 40 replicates of prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. Twenty replicates of spiked hemoglobin-negative stool concentrations were collected without toilet water and 20 replicates of spiked hemoglobin-negative stool concentrations were collected submerged with toilet water. All samples were mixed with detection buffer in Instant-view-plus™ Immunochemical Fecal Occult Blood Test sample collection bottles and tested in a randomized order.

Assay cut-off: Fecal test samples were prepared by spiking stool samples with human blood of known hemoglobin concentration, to obtain the following fecal hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 48 ng/ml, 60 ng/ml, 60 ng/ml, 72 ng/ml and 500 ng/ml. Twenty aliquots of each of the seven concentrations of spiked stool test samples were tested in randomized order. Testing was performed side-by-side with the predicate by comparing the test results of the device with that of the predicate.

Method comparison with predicate device: A method comparison of Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test with the predicate test, Instant-view™ Fecal Occult Blood Rapid Test, was conducted by assessing 299 patient samples. The method comparison study was performed at three POC testing sites by two operators at each site. The Insure® FITTM FOBT Controls (K101831) Negative and Positive Controls were run prior to testing.

Consumer Study: A consumer study was performed by testing Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test with the predicate test, Instantview® Fecal Occult Blood (FOB) Self-Test hemoglobin free feces spiked samples at the following concentrations: 0 ng/ml, 25 ng/ml, 50 ng/ml, 60 ng/ml, and 500 ng/ml.

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

Precision/Reproducibility:
Repeatability:
Lot 1: Positive Percent Agreement 100% (164/164) (97.8%, 100%), Negative Percent Agreement 100% (136/136) (97.3%, 100%)
Lot 2: Positive Percent Agreement 100% (164/164) (97.8%, 100%), Negative Percent Agreement 97.8% (133/136) (93.7%, 99.5%)
Lot 3: Positive Percent Agreement 100% (164/164) (97.8%, 100%), Negative Percent Agreement 99.3% (135/136) (96.0%, 99.9%)
Combined Lots: Positive Percent Agreement 100% (492/492) (99.3%, 100%), Negative Percent Agreement 99% (404/408) (97.5%, 99.7%)

Reproducibility - Lot Variability:
Lot 1: Positive Percent Agreement 100 % (684/684) (99.5%, 100%), Negative Percent Agreement 98.6% (568/576) (97.3%, 99.4%)
Lot 2: Positive Percent Agreement 100 % (684/684) (99.5%, 100%), Negative Percent Agreement 97% (559/576) (95.3%,98.3%)
Lot 3: Positive Percent Agreement 100 % (684/684) (99.5%, 100%), Negative Percent Agreement 96.4% (555/576) (94.5%, 97.7%)

Reproducibility - Day Variability:
Day 1: Positive Percent Agreement 100% (410/410) (99.1%, 100%), Negative Percent Agreement 95.1% (329/346) (92.3%, 97.1%)
Day 2: Positive Percent Agreement 100% (410/410) (99.1%, 100%), Negative Percent Agreement 89% (308/346) (85.2%, 92.1%)
Day 3: Positive Percent Agreement 100% (410/410) (99.1%, 100%), Negative Percent Agreement 98.6% (341/346) (96.7%, 99.5%)
Day 4: Positive Percent Agreement 100% (410/410) (99.1%, 100%), Negative Percent Agreement 95.4% (330/346) (92.6%, 97.3%)
Day 5: Positive Percent Agreement 100% (410/410) (99.1%, 100%), Negative Percent Agreement 96.8% (335/346) (94.4%, 98.4%)

Reproducibility - Site Variability:
Site 1: Positive Percent Agreement 100% (684/684) (99.5%, 100%), Negative Percent Agreement 97.1% (559/576) (95.3%, 98.3%)
Site 2: Positive Percent Agreement 100% (684/684) (99.5%, 100%), Negative Percent Agreement 97.4% (561/576) (95.7%, 98.5%)
Site 3: Positive Percent Agreement 100% (684/684) (99.5%, 100%), Negative Percent Agreement 96.7% (557/576) (94.9%, 98%)

Prozone Effect Study: The results show that Instant-view-plus™ Immunochemical Fecal Occult Blood Test was not affected by highly concentrated hemoglobin in specimen, no prozone effect was observed in the above Hb concentrations.

Stability Studies:
Test Kit Stability (Accelerated): The test kit was determined to be stable for 24 months, when stored at the temperatures between 8 - 23°C (46.4 - 73.4°F).
Test Kit Stability (Real Time): The test kit was determined to be stable for at 24 months.

Analytical specificity:
Specificity to human hemoglobin variant: Results showed Instant-view-plus™ Immunochemical Fecal Occult Blood Test equivalently recognizes variants of hemoglobin HbA, HbS and HbC.
Cross-Reactivity: Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant cross-reactivity with any of the animal hemoglobins tested.
Interfering Substances: Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant interference from any of the vegetable extracts.
Interfering Supplements: Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant interference from iron and sodium L-ascorbate.
Interference from Toilet Water: Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant interference from samples collected in toilet water.

Assay cut-off: The cut-off was determined to be 50 ng/ml (hemoglobin in fecal sample mixed with detection buffer).
Method comparison with predicate device: The method comparison study demonstrated that the analytical performance of the Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test is substantially equivalent to the predicate device.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Not Found

Predicate Device(s)

K070660, K021423

Reference Device(s)

K101831

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

§ 864.6550 Occult blood test.

(a)
Identification. An occult blood test is a device used to detect occult blood in urine or feces. (Occult blood is blood present in such small quantities that it can be detected only by chemical tests of suspected material, or by microscopic or spectroscopic examination.)(b)
Classification. Class II (special controls). A control intended for use with an occult blood test is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 864.9.

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Image /page/0/Picture/0 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which consists of the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG" in blue, and then the word "ADMINISTRATION" in a smaller font size below that.

Alfa Scientific Designs, Inc. Wen Hua Fan RA Specialist 13200 Gregg St Poway, California 92064

February 15, 2018

Re: K173212

Trade/Device Name: Instant-view-plus immunochemical Fecal Occult Blood Test Regulation Number: 21 CFR 864.6550 Regulation Name: Occult blood test Regulatory Class: Class II Product Code: KHE Dated: January 11, 2018 Received: January 16, 2018

Dear Wen Hua Fan:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

1

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Kelly Oliner -S

For,

Lea Carrington Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K173212

Device Name

Instant-view-plus™ Immunochemical Fecal Occult Blood Test

Indications for Use (Describe)

The Instant-view-plus™ Immunochemical Fecal Occult Blood Test is a qualitative immunoassay for detection of Fecal Occult Blood. It is intended for professional and over the counter use.

Type of Use (Select one or both, as applicable)
☒ Prescription Use (Part 21 CFR 801 Subpart D)☒ Over-The-Counter Use (21 CER 801 Subpart C)

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510(k) Summary

Safety and Effectiveness as Required by 21 CFR 807.92
Date Prepared:February 8, 2018
Manufacture and Submitter:Alfa Scientific Designs, Inc.
13200 Gregg Street
Poway, CA 92064
Telephone (858) 513-3888 x 324
Fax: (858) 513-8388
Contact Person: Wen Hua Fan, Ph.D.
E-mail: wfan@alfascientific.com
Device Name:Trade Name : Instant-view plusTM Immunochemical
Fecal Occult Blood Test
Common Name: Fecal Immunochemical Test
Classification Name: Occult Blood Test
Classification: Class II
Product Code: KHE
Predicate Device:K070660 and K021423

Device Description: This device is a Driven Flow™ chromatographic immunoassay consisting of a test strip housed in a plastic cassette.

.

4

Similarities
ItemDevice
Instant-view-plusTM
Immunochemical
Fecal Occult Blood
Test, K173212Predicate
Instant-view®Fecal
Occult Blood (FOB)
Self-Test
K070660Predicate
Instant-view™ Fecal
Occult Blood Rapid
Test
K021423
Intended useThe Instant-view-
plus™
Immunochemical
Fecal Occult Blood
Test is a qualitative
immunoassay for
detection of Fecal
Occult Blood. It is
intended for
professional and over
the counter use.The Instant-view®
Fecal Occult Blood
(FOB) Rapid Test is a
qualitative
immunoassay for the
detection of Fecal
Occult Blood by non-
professional, lay
individuals as an over-
the-counter product
for home use.
Measurement of FOB
is a useful as an aid to
detect blood in stool
as found in a number
of gastrointestinal
disorders, e.g.
diverticulitis, colitis,
polyps, and colorectal
cancer. It is intended
for over-the-counter
use. The Fecal Occult
Blood (FOB) Rapid
Test is a qualitative
immunoassay for the
detection of Fecal
Occult Blood by non-
professional, lay
individuals as an over-
the-counter product
for home use.
Measurement of FOB
is a useful as an aid to
detect blood in stool
as found in a number
of gastrointestinal
disorders, e.g.
diverticulitis, colitis,This Fecal Occult
Blood (FOB) Rapid
Test is an
immunochemical
device intended for
the qualitative
detection of Fecal
Occult Blood by
laboratories or
physicians' offices.
It is useful to
determining
gastrointestinal (GI)
bleeding found in a
number of
gastrointestinal (GI)
disorders, e.g.,
diverticulitis, colitis,
polyps, and
colorectal cancer.
This test is
recommended for
use in 1) routine
physical
examinations, when
hospital patients are
first admitted, 2)
hospital monitoring
for bleeding in
patients, 3)
screening for
colorectal cancer or
gastrointestinal
bleeding & any
source.
Similarities
ItemDevice
Instant-view-plusTM
Immunochemical
Fecal Occult Blood
Test, K173212Predicate
Instant-view®Fecal
Occult Blood (FOB)
Self-Test
K070660Predicate
Instant-viewTM Fecal
Occult Blood Rapid
Test
K021423
polyps, and colorectal
cancer. It is intended
for over-the-counter
use.
Sample typeHuman feces
(mixed with detection
buffer)SameSame
Assay cut-off50 ng/ml (human
hemoglobin in human
fecal sample in
extraction buffer)SameSame

Device Characteristics in Comparison with the Predicate Devices:

5

Differences
ItemDevice
Instant-view-plus™
Immunochemical Fecal
Occult Blood Test,
K173212Predicate
Instant-view® Fecal
Occult Blood (FOB)
Self-Test
K070660Predicate
Instant-view™
Fecal Occult Blood
Rapid Test
K021423
Test principleChromatographic
immunoassay using
Driven Flow™
TechnologyLateral flow
chromatographic
immunoassayLateral flow
chromatographic
immunoassay
Test deviceCassetteCassette and dip stripCassette and dip strip
Assay Reading
TimeBetween 1-10 minutes4-7 minutes5-10 minutes

Test Principle:

This assay is a Driven Flow chromatographic immunoassay. The device consists of one test strip in a plastic cassette. The test strip consists of:

  • . A burgundy colored conjugate pad treated with mouse anti-hHb antibodies conjugated with colloidal gold.

6

  • A strip of nitrocellulose membrane with a Test line (T-line) and a Control line . (C-line). The T-line is coated with anti-hHb antibodies, and the C-line is coated with goat anti-mouse IgG antibodies.
    When an adequate volume of test specimen is dispensed into the sample well of the device, the test specimen migrates across the test strip. If the concentration of hHb in the specimen is at or above 50 ng/ml, the T-line appears as a visible burgundy line. The intensity of the T-line may vary according to the concentration of the hHB in the samples. If the concentration of hHb in the specimen is below the detectable level, no T-line develops. The C-line is coated with goat anti-mouse antibody, which binds to the conjugated monoclonal antibody, regardless of the presence of hHb in the sample.

Precision/Reproducibility:

Repeatability was evaluated using three lots of devices, in-house by two trained laboratory professionals. For repeatability Hb-free fecal samples were collected and spiked with human whole blood with known hemoglobin levels to achieve the following six fecal hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 50 ng/ml, 55 ng/ml, and 500 ng/ml. Fifty replicates were performed for each concentration level.

| | Positive Percent
Agreement
(#Positive/#Total)
(95% CI) | Negative Percent
Agreement
(#Negative/#Total)
(95% CI) |
|------------------|-----------------------------------------------------------------|-----------------------------------------------------------------|
| Lot 1 | 100% (164/164)
(97.8%, 100%) | 100% (136/136)
(97.3%, 100%) |
| Lot 2 | 100% (164/164)
(97.8%, 100%) | 97.8% (133/136)
(93.7%, 99.5%) |
| Lot 3 | 100% (164/164)
(97.8%, 100%) | 99.3% (135/136)
(96.0%, 99.9%) |
| Combined
Lots | 100% (492/492)
(99.3%, 100%) | 99% (404/408)
(97.5%, 99.7%) |

Reproducibility was conducted across three intended use sites using three device lots (same three lots at all sites), and three operators (one at each site) over a minimum of 5 days. For reproducibility Hb-free fecal samples were collected and spiked with human whole blood with known hemoglobin levels to achieve the following nine fecal hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 50 ng/ml, 60 ng/ml, 60 ng/ml, and 500 ng/ml. Fourteen replicates were performed for each sample and concentration level. Insure® FITTM FOBT Controls (K101831) Negative and Positive Controls were also tested daily to ensure the validity of the test results.

Reproducibility - Lot Variability

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| Lot | Positive Percent Agreement
(#Positive/#Total)
(95% CI) | Negative Percent Agreement
(#Negative/#Total)
(95% CI) |
|-------|--------------------------------------------------------------|--------------------------------------------------------------|
| Lot 1 | 100 % (684/684)
(99.5%, 100%) | 98.6% (568/576)
(97.3%, 99.4%) |
| Lot 2 | 100 % (684/684)
(99.5%, 100%) | 97% (559/576)
(95.3%,98.3%) |
| Lot 3 | 100 % (684/684)
(99.5%, 100%) | 96.4% (555/576)
(94.5%, 97.7%) |

Reproducibility - Day Variability

| Day | Positive Percent Agreement
(#Positive/#Total)
(95% CI) | Negative Percent Agreement
(#Negative/#Total)
(95% CI) |
|-------|--------------------------------------------------------------|--------------------------------------------------------------|
| Day 1 | 100% (410/410)
(99.1%, 100%) | 95.1% (329/346)
(92.3%, 97.1%) |
| Day 2 | 100% (410/410)
(99.1%, 100%) | 89% (308/346)
(85.2%, 92.1%) |
| Day 3 | 100% (410/410)
(99.1%, 100%) | 98.6% (341/346)
(96.7%, 99.5%) |
| Day 4 | 100% (410/410)
(99.1%, 100%) | 95.4% (330/346)
(92.6%, 97.3%) |
| Day 5 | 100% (410/410)
(99.1%, 100%) | 96.8% (335/346)
(94.4%, 98.4%) |

Reproducibility - Site Variability

| Site | Positive Percent Agreement
(#Positive/#Total)
(95% CI) | Negative Percent Agreement
(#Negative/#Total)
(95% CI) |
|--------|--------------------------------------------------------------|--------------------------------------------------------------|
| Site 1 | 100% (684/684)
(99.5%, 100%) | 97.1% (559/576)
(95.3%, 98.3%) |
| Site 2 | 100% (684/684)
(99.5%, 100%) | 97.4% (561/576)
(95.7%, 98.5%) |
| Site 3 | 100% (684/684)
(99.5%, 100%) | 96.7% (557/576)
(94.9%, 98%) |

Prozone Effect Study:

The Prozone effect was evaluated using three device lots, by two operators testing Hbfree stool specimens spiked with human blood of known hemoglobin concentrations so as to obtain fecal test samples with different Hb concentrations: 1,000 ng/ml (1 ug/ml),

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2,000 ng/ml (2 ug/ml), 3,000 ng/ml (3 ug/ml), 4,000 ng/ml (4 ug/ml), 5,000ng/mL (5 ug/ml), 50,000 ng/mL (50 ug/ml), and 500,000 ng/mL (500 ug/ml). Twenty aliquots of each sample concentration were mixed with extraction buffer in the specimen collection tubes and tested in a randomized order. The results show that Instant-view-plus"M Immunochemical Fecal Occult Blood Test was not affected by highly concentrated hemoglobin in specimen, no prozone effect was observed in the above Hb concentrations.

Stability Studies:

Insure® FIT™ FOBT Controls (K101831) Negative and Positive Controls were also tested daily to ensure and confirm the validity of the test results of the stability studies.

    1. Test Kit Stability (Accelerated)
      The accelerated stability study was conducted with three lots of Instant-view-plus™ Immunochemical Fecal Occult Blood Test. Hb-free stool specimens were spiked with human blood (of known hemoglobin level) to obtain fecal samples containing six different hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. Each hemoglobin concentration was tested with two devices from each lot. (2) operator tested the three lots stored at following four temperatures: 20°C (68°F), 23°C (73.4°F), 35°C (95°F), and 60°C (140°F) and test results were collected each day for 20 days. The test kit was determined to be stable for 24 months, when stored at the temperatures between 8 - 23°C (46.4 - 73.4°F).
    1. Test Kit Stability (Real Time)
      The real time stability study was conducted with three lots of Instant-view-plus™ Immunochemical Fecal Occult Blood Test. Hb-free stool specimens were spiked with human blood (of known hemoglobin level) to obtain fecal samples containing six different hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 60 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. Each hemoglobin concentration was tested with four devices from each lot. (2) operators tested the three lots stored at room temperature (20 ~ 23°C) for 26 months. The test kit was determined to be stable for at 24 months.

Analytical specificity:

Insure® FITTM FOBT Controls (K101831) Negative and Positive Controls were also tested daily to ensure the validity of the test results.

Specificity to human hemoglobin variant

The ability of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to detect human hemoglobin variants was determined by testing hemoglobin-A (HbA). hemoglobin-S (HbS) and hemoglobin-C (HbC) by preparing spiked stool samples containing seven different hemoglobin concentrations: 0 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. (3) aliquots of each of the seven concentrations (7 HbA, 7 HbS and 7 HbC supernatants) of spiked stool test samples were

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mixed with detection buffer in Instant-view-plus™ Immunochemical Fecal Occult Blood Test collection bottles. Samples were tested in a randomized order. Results showed Instant-view-plus™ Immunochemical Fecal Occult Blood Test equivalently recognizes variants of hemoglobin HbA, HbS and HbC.

Cross-Reactivity

Cross-reactivity of Instant-view-plus™ Immunochemical Fecal Occult Blood Test with animal hemoglobin was evaluated by testing prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. Five replicates of spiked hemoglobin-negative stool concentrations listed above were then spiked with the intended level of respective animal hemoglobin such as beef (2000 ug/ml), chicken (500 ug/ml), fish (100 µg/ml), horse (500 ug/ml), goat (500 µg/ml), rabbit (500 µg/ml), pig (500 µg/ml), horseradish peroxidase (20.000 ug/ml), and sheep (100 ug/ml). All samples were tested in a randomized order. Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant cross-reactivity with any of the animal hemoglobins tested.

Interfering Substances

Susceptibility of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to interference from extracts (vegetable) was evaluated by testing (5) replicates of prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml, 48 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. The 5 replicates of the spiked hemoglobin-negative stool concentrations listed above were spiked with the intended level of respective vegetable extracts: broccoli extracts (100%), cantaloupe extract (100%), cauliflower extract (100%), parsnip extract (100%), red radish extract (100%), and turnip extract (100%). All samples were mixed with detection buffer in Instant-viewplus™ Immunochemical Fecal Occult Blood Test sample collection bottles and tested in a randomized order. Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant interference from any of the vegetable extracts.

Interfering Supplements

Susceptibility of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to interference from iron and sodium L-ascorbate was evaluated by testing 3 replicates of prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml. 48 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml. The 3 replicates of spiked hemoglobin-negative stool concentrations were spiked with iron (0.5% (w/v), and sodium L-ascorbate (0.5% (w/v). All samples were mixed with detection buffer in Instant-viewplus™ Immunochemical Fecal Occult Blood Test sample collection bottles and tested in a randomized order. Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant interference from iron and sodium L-ascorbate.

Interference from Toilet Water

Susceptibility of Instant-view-plus™ Immunochemical Fecal Occult Blood Test to

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interference from toilet water was evaluated by testing 40 replicates of prepared test samples by spiking Hb-free stool specimen with known levels of human Hb solution to obtain fecal samples with seven different Hb concentrations: 0 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 72 ng/ml, and 500 ng/ml. Twenty replicates of spiked hemoglobinnegative stool concentrations were collected without toilet water and 20 replicates of spiked hemoglobin-negative stool concentrations were collected submerged with toilet water. All samples were mixed with detection buffer in Instant-view-plus™ Immunochemical Fecal Occult Blood Test sample collection bottles and tested in a randomized order. Instant-view-plus™ Immunochemical Fecal Occult Blood Test did not show significant interference from samples collected in toilet water.

Assay cut-off:

The cut-off value for the Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test was validated in-house. Fecal test samples were prepared by spiking stool samples with human blood of known hemoglobin concentration, to obtain the following fecal hemoglobin concentrations: 0 ng/ml, 25 ng/ml, 48 ng/ml, 60 ng/ml, 60 ng/ml, 72 ng/ml and 500 ng/ml. Twenty aliquots of each of the seven concentrations of spiked stool test samples were tested in randomized order. Testing was performed side-by-side with the predicate by comparing the test results of the device with that of the predicate. The cut-off was determined to be 50 ng/ml (hemoglobin in fecal sample mixed with detection buffer).

| Concentration | N | Instant-view-plus™™
plus
Immunochemical
Fecal Occult Blood
Test | | Percent Positive | Percent Negative |
|---------------|----|-----------------------------------------------------------------------------|----------|-----------------------|-------------------------|
| | | Positive | Negative | | |
| 0 ng/ml | 20 | 0 | 20 | 0%
0% - 15.5% | 100.00%
84.5% - 100% |
| 25 ng/ml | 20 | 1 | 19 | 5%
0.80% - 19.80% | 95%
80.20% - 99.20% |
| 48 ng/ml | 20 | 11 | 9 | 55%
39.12% - 70.82 | 45%
29.18% - 60.82% |
| 50 ng/ml | 20 | 11 | 9 | 55%
39.12% - 70.82 | 45%
29.18% - 60.82% |
| 60 ng/ml | 20 | 20 | 0 | 100%
84.5% - 100% | 0%
0% - 15.5% |
| 72 ng/ml | 20 | 20 | 0 | 100%
84.5% - 100% | 0%
0% - 15.5% |
| 500 ng/ml | 20 | 20 | 0 | 100%
84.5% - 100% | 0%
0% - 15.5% |

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Method comparison with predicate device:

A method comparison of Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test with the predicate test, Instant-view™ Fecal Occult Blood Rapid Test, was conducted by assessing 299 patient samples. The method comparison study was performed at three POC testing sites by two 2 operators at each site. The Insure® FITTM FOBT Controls (K101831) Negative and Positive Controls were run prior to testing. Statistical analysis of site-wide test results as well as combined results showed that Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test results have acceptable overall percent agreement as well as positive percent agreement and negative percent agreement with Instant-view™ Fecal Occult Blood Rapid Test. The method comparison study demonstrated that the analytical performance of the Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test is substantially equivalent to the predicate device.

| Study Site | Instant-view-
plusTM
Immunochemical
FOB Test | Predicate Test
Instant-View Fecal Occult
Blood Rapid Test | | | Negative
Percent
Agreement
(95% CI) | Positive Percent
Agreement
(95% CI) | Overall Percent
Agreement
(95% CI) |
|-------------------|-------------------------------------------------------|-----------------------------------------------------------------|----------|-------|----------------------------------------------|-------------------------------------------|------------------------------------------|
| | | Positive | Negative | Total | | | |
| Site 1 | Positive | 36 | 1 | 37 | 98.5%
(92.1%, 99.9%) | 94.7%
(82.3%, 99.4%) | 97.2%
(92.0%, 99.4%) |
| | Negative | 2 | 67 | 69 | | | |
| | Total | 38 | 68 | 106 | | | |
| Site 2 | Positive | 36 | 1 | 37 | 98.9%
(93.8%, 99.9%) | 97.3%
(85.8%, 99.9%) | 98.4%
(94.3%, 99.8%) |
| | Negative | 1 | 87 | 88 | | | |
| | Total | 37 | 88 | 125 | | | |
| Site 3 | Positive | 25 | 1 | 26 | 97.6%
(87.4%, 99.9%) | 96.2%
(80.4%, 99.9%) | 97.1%
(89.8%, 99.6%) |
| | Negative | 1 | 41 | 42 | | | |
| | Total | 26 | 42 | 68 | | | |
| Combined
Sites | Positive | 97 | 3 | 100 | 98.5%
(95.6%, 99.7%) | 6.0%
(90.2%, 98.9%) | 97.7%
(95.2%, 99.1%) |
| | Negative | 4 | 195 | 199 | | | |
| | Total | 101 | 198 | 299 | | | |

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Consumer Study:

To support over the counter use, a consumer study was performed by testing Instant-view plus™ Immunochemical Fecal Occult Blood (FOB) Test with the predicate test, Instantview® Fecal Occult Blood (FOB) Self-Test hemoglobin free feces spiked samples at the following concentrations: 0 ng/ml, 25 ng/ml, 50 ng/ml, 60 ng/ml, and 500 ng/ml.

| Concentration | Number
of
Samples | Predicate Device | | New Device | | Difference (New - Predicate) | |
|---------------|-------------------------|------------------|-----------------|-----------------|-----------------|-----------------------------------------|-----------------------------------------|
| (ng/mL) | | Negative
(%) | Positive
(%) | Negative
(%) | Positive
(%) | Negative %
(95% CI of
Difference) | Positive %
(95% CI of
Difference) |
| 0 | રત | રવ | 0 | રવ | 0 | 0
(-6.8%,6.8%) | 0
(-6.8%,6.8%) |
| 25 | રત | 52 | 2 | રે રે | 1 | 1.9%
(-6.8%, 1.1%) | -1.9%
(-11.1%, 6.8%) |
| 50 | રત | 25 | 29 | 26 | 28 | 1.9%
(-17.4%, 21.0%) | -1.9%
(-21.0%, 17.4%) |
| 60 | રત | l | રે રે | l | રે રે | 0
(-8.6%, 8.6%) | 0
(-8.6%, 8.6%) |
| 500 | રત | 0 | ર્ડવ | 0 | રવ | 0
(-6.8%, 6.8%) | 0
(-6.8%,6.8%) |