SUCCEED Equine Fecal Blood Test

4. Add clean (potable) tap water to the fecal matter in the container. Fill to the Water Fill Line (top line). Do not use water from dirty containers or from sources that may be contaminated with equine blood. Water should be room temperature.

5. Replace cap on container, and shake vigorously to mix fecal matter and water. Set container on a flat surface, and remove lid.

6. Open foil pack and remove test cassette and sample pipette. (Discard empty foil pack and desiccant pouch.)

7. Use provided disposable pipette to collect solution from container. Collect liquid only, minimizing particles. For best results: (i) tip jar slightly, (ii) depress pipette bulb before placing in solution, (iii) insert tip of pipette below surface of solution and release bulb slowly to draw up solution.

8. Apply 2 drops of solution from pipette to EACH of the two sample wells. Depress pipette bulb slowly to produce large drops.

Watch for liquid to wick up the membrane in the test window: it should wick to above the CTL line in 2-3 minutes. If wicking does not occur, check that well is not clogged with silt, and apply one more drop ONLY to non-wicking well. (Excess fluid can flood the test, negating the result.)

9. Read results. A positive result may appear within 5 minutes after solution has clearly wicked up test membrane, but may take longer. DO NOT read results after 15 minutes. If a line appears at the TST point on either the Test A or Test H window, that is a positive test. Note that even a faint line reflects a positive test.

Failure to carefully follow these instructions may lead to incorrect interpretation.

Reading The Tests

Observe results after applying the solution to each of the two sample wells on the cassette, per instructions.

1. If a Control line does not appear within 15 minutes (at the CTL point on the cassette) that test result is invalid. The test must then be repeated with a new SUCCEED Equine Fecal Blood Test.

2. If only a Control line appears on either strip, that test is NEGATIVE. NOTE: only a complete line is a meaningful result.

If a solid line appears only at the CTL point (Control line) on either Test A or Test H, this is a NEGATIVE result.

3. If both the Control and Test lines appear on either window (Test A or H), this indicates a POSITIVE test result. Even if the test line is faint, the test is POSITIVE.

If a complete, solid line appears at the TST point (Test line) on either or both Test A and Test H, that is a POSITIVE test result. Test A detects equine albumin. Test H detects equine hemoglobin.

Note - even if the test line is faint, the test is POSITIVE.

4. Do not read test results after 15 minutes.

If you have questions about reading the SUCCEED Equine Fecal Blood Test, please call: 800-281-6727 from 8AM - 5PM ET Monday through Friday.

Interpreting The Results

The following chart summarizes the meaning of a positive and/or negative test results across SUCCEED FBT tests A and H.

* Note that Test A uses an antibody for albumin, which typically breaks down in digestion, particularly when exposed to digestive enzymes in the proximal portion of the small intestine. Thus a positive Test A will indicate bleeding from a source caudal to the duodenum (e.g. hindgut). Also, because albumin may be present at the site of low-grade injury absent whole blood, you may get a positive Test A in response to a low-grade lesion in the hindgut, such as may occur with heavy parasitism or disseminated grade 1 colonic ulcers.

Test H uses an antibody for hemoglobin, which is more resistant to enzymatic activity. Thus a positive Test H, with negative Test A, will indicate occult blood from a foregut source. You will not obtain a positive result from either Test A or H when a horse has a foregut lesion equivalent to a grade 1 gastric ulcer, where whole blood loss does not occur.

Limitations Of The Test

The SUCCEED FBT is a fecal occult blood test. It is designed to detect blood components in feces that may be indicative of underlying pathologic conditions. This makes the SUCCEED FBT a qualitative test for the presence and location of gastrointestinal injury. It is not a quantitative test.

Albumin may be present at the site of injury absent whole blood. Thus a positive test A reflects injury equivalent to grade 1 or higher ulceration in the hindgut. Hemoglobin is only present with whole blood. Therefore, a positive test H reflects injury equivalent to grade 2 or higher ulceration anywhere in the GI tract.

Equine albumin and hemoglobin in feces may arise from a variety of conditions, including but not limited to:

- Bleeding ulcers (gastric or colonic)

- Excessive parasitic activity

- Protein-losing enteropathy (PLE)

- Lesions caused by hard particles of feed or other ingested material

- Petechiation, hyperemias or lypomas

Equine GI lesions - including petechiation and parasite pits - may bleed lightly or intermittently, so a test result may be negative even when disease is present. And because blood components may be non-uniformly distributed in a fecal dropping, negative readings may occasionally be obtained from a fecal sample of an unhealthy horse. As with any fecal blood test, SUCCEED FBT is not a conclusive diagnostic for occult gastrointestinal bleeding or pathology; it should be employed only as an aid to diagnosis.

Additionally, a positive test result (especially Test H), can arise from extraneous sources, such as:

- Dentistry or exercise-induced pulmonary hemorrhage

- Mare ovulation

- Post-surgery bleeding

As with any diagnostic, a definitive diagnosis should not be based on the result of a single test. The test is designed to be complementary to blood tests and physical examination. NOTE: Practitioners experiencing a positive Test A without a positive Test H, should include a protein-losing enteropathy as one differential diagnosis, especially in the presence of hypoproteinemia/hypoalbuminemia on a CBC/chem profile. A negative test result in conjunction with hypoproteinemia/hypoalbuminemia may reflect a protein-losing nephropathy.

Applications

Because symptoms are an unreliable indicator of GI health, practitioners should test horses periodically. Given the ease and affordability of the FBT, testing all horses on a regular schedule is a practical option for early detection.

Consistent testing is especially important for performance horses, or wherever the care, feeding and general husbandry can affect digestive health, including intermittent feeding, high-grain diets, stall confinement, etc. Because it is possible that GI health may be a root cause of many performance or behavioral problems, horses with such issues may be prime candidates for testing.

The SUCCEED FBT may also be a valuable part of a pre-purchase exam.

Performance Characteristics

The performance of the SUCCEED FBT was assessed with multiple in vitro and in vivo tests, with the results as detailed below.

Analytical Sensitivity

The antibodies used in the two parts of the SUCCEED FBT - Test A and Test H - were evaluated by introducing equine blood at varying concentrations into a solution of water and rabbit fecal matter. The detection limits of the FBT antibodies, at a 95% confidence level, are provided here.

Fig 1: Detection limits of FBT antibodies

Diagnostic Reliability

Figure 2 indicates the statistical accuracy of the SUCCEED FBT results as a reflection of actual pathological conditions in horses, as determined by comparing FBT results on fecal samples from equine subjects with observed GI tract injury in those same subjects post-necropsy.

The SUCCEED FBT has been calibrated to detect blood components at levels reflecting true pathologies and to ignore subclinical or baseline bleeding.

Fig 2: Analysis of tests A & H as indicators of ulceration

SUCCEED^® and Digestive Conditioning Program^® are registered trademarks, and Equine Fecal Blood Test™ is a trademarks of Freedom Health LLC ©2011, 2012. All Rights Reserved.

SUCCEED^® Digestive Conditioning Program® holds U.S. Patent Nos. 7,658,964; 7,824,706 and 7,988,989. Further patents pending.

SUCCEED^® Equine Fecal Blood Test™ holds U.S. Patent No. 7,629,180.

Contact Information:

Freedom Health, LLC, 65 Aurora Industrial Parkway, Aurora, OH 44202

Toll-free service line: 877-734-6558 24 hours a day

For more information, visit our website at www.SucceedFBT.com

Also available from Freedom Health:

SUCCEED^® Digestive Conditioning Program^® Veterinary Formula.

I501 Rev H 04/12

Nac No.