Witness HW

Canine Heartworm Antigen Test Kit

For the detection of canine and feline heartworm infection

I. Heartworm Disease

Heartworm disease is caused by Dirofilaria immitis, a filarial nematode whose immature stages are transmitted between animals by mosquitoes. In dogs, the adult heartworms live in the heart and adjacent large blood vessels. In cats, heartworms affect primarily the lungs and lead to Heartworm Associated Respiratory Disease (HARD). Although primarily a disease of dogs, the parasite can also infect ferrets and other mammals, such as the red fox

and the coyote.

Ii. Test Principles

The WITNESS^® HW test is a simple test, based on rapid immunomigration (RIM) technology, which detects the presence of adult D. immitis antigens in dog or cat blood. Sensitized particles bound to the heartworm antigens present within the sample (whole blood, serum or plasma) migrate along a nitrocellulose strip. The complex is then captured on a sensitized reaction zone where its accumulation causes the formation of a clearly visible pink/purple band. A pink/purple band in the control window ensures that the test was performed correctly.

Iii. Sample Information

50uL (0.05 mL) of anticoagulated (EDTA or heparin) whole blood, plasma or serum is required. Hemolysis does not significantly interfere with the test, but strongly hemolyzed samples may partly obscure a weak positive band.

Iv. Sample Storage

Fresh, anticoagulated whole blood should be tested within 4 hours. Anticoagulated whole blood may be stored at refrigeration for up to 24 hours. If longer storage is required, serum or plasma may be stored at refrigeration (2° to 7°C; 35° to 45°F) for up to 7 days. Serum or plasma samples may be frozen (-20°C) if even longer storage is required.

V. Kit Contents

- 10 or 25 pouches, each containing 1 test device.

- 10 or 25 disposable pipettes.

- 1 buffer dropper bottle (5.0 mL).

- Instructions for use.

Vi. Precautions

- Do not use components after expiration date.

- Refrigeration not required. Store the test kit at 2° to 25°C (35° to 77°F). Do not freeze.

- Use the test within 30 minutes after opening the sealed pouch.

- Avoid touching or damaging membrane at windows #1, #2, #3.

- The WITNESS^® device should be placed on a flat, horizontal surface while performing the test.

- Use a separate pipette for each sample.

- Hold pipette and buffer bottle vertically when dispensing sample and buffer.

- Do not mix components of different serials.

- For veterinary use only.

Vii. Test Procedure And Results

Important: Allow sample and buffer drops to fall onto membrane at window #1. Do not touch pipette tip, sample or buffer drops, or buffer bottle tip directly to the membrane.

1. Sample Application

- Tear open the pouch provided and place the test device on a flat horizontal surface for the duration of the test.

- Squeeze the provided pipette near the sealed end. Insert the open end of the pipette into an anticoagulated whole blood, serum, or plasma sample. Release the pressure slightly to draw up a small amount of sample into the pipette.

- Holding the pipette vertically, transfer one drop of sample to the sample well, window #1. Allow sample to be completely absorbed.

2. Buffer Dispensing

- Remove the cap from the buffer bottle, hold it vertically and add two drops of buffer to the sample well window #1.

3. Reading Test

- Wait ten minutes, observe the presence or absence of pink/purple bands in reading windows #2 and #3.

- Sample results are read in window #2. The control band is read in window #3.

Notes: - The test is complete and may be read before 10 minutes if pink/purple bands are visible in both windows #2 and #3.

- The presence of a pink/purple band only in window #3 before 10 minutes does not mean that the test is complete. A pink/purple band in window #2 may develop slower than the control pink/purple band in window #3.

4. Results

Validation

- Valid test: Test is validated if a pink/purple band is present in the reading window #3.

Interpretation

- Negative for HW antigen: No band in reading window #2, with one pink/purple band in window #3.

- Positive for HW antigen: One pink/purple band in reading window #2, with one pink/purple band in window #3.

Invalid Test

- Invalid test: No pink/purple band in control window #3.

Note: A test result should always be interpreted in the context of all available clinical information and history for the dog or cat. Cats testing negative for heartworm antigen may benefit from other diagnostic tests, including heartworm antibody assays.

Witness^® Test Procedure

Witness® Hw Test Performance Comparison

Study Id	Sample Size kit/reference				Total	Sample Type	Statistical Test	Sensitivity & Specificity 95% Confidence Limit (ci)
	+/+	-/+	+/-	-/-
1a	48	2	0	50	100	Serum	Binomial Distribution	Sen: 96% [95% CI: 86.5% - 98.9%] Spec: 100% [95% CI: 93% - 100%]
1b	5	0	0	36	41	Whole Blood EDTA	Binomial Distribution	Sen: 100% [95% CI: 56.6% - 100%] Spec: 100% [95% CI: 90.4% - 100%]
	1	0	0	36	37	Whole Blood Heparin	Binomial Distribution	Sen: 100% [95% CI: 20.7% - 100%] Spec: 100% [95% CI: 90.4% - 100%]
1c	5	0	0	36	41	Plasma EDTA	Binomial Distribution	Sen: 100% [95% CI: 56.6% - 100%] Spec: 100% [95% CI: 90.4% - 100%]
	1	0	0	36	37	Plasma Heparin	Binomial Distribution	Sen: 100% [95% CI: 20.7% - 100%] Spec: 100% [95% CI: 90.4% - 100%]
2	98	2	0	30	130	Serum	Binomial Distribution	Sen: 98% [95% CI: 93.0% - 99.5%] Spec: 100% [95% CI: 88.7% - 100%]
Cumulative (Studies #1 and #2)
	158	4	0	224	386	All	Binomial Distribution	Sen: 97.5% [95% CI: 93.8% - 99.0%] Spec: 100% [95% CI: 98.3% - 100%]

Study #1 - Results from WITNESS^® HW Efficacy Verification Study, May 16, 2006

1^a Kit results/Reference Test Results (Serum Samples characterized by Necropsy)

1^b Kit results/Reference Test Results (Whole Blood Samples in Heparin and EDTA characterized by ELISA)

1^c Kit results/Reference Test Results (Plasma Samples in Heparin and EDTA characterized by ELISA)

Study #2 - Results from WITNESS® HW Serology Study, April 13, 2007

Kit results/Reference Test Results (Serum Samples characterized by Necropsy)

Ordering Information

Manufactured By

Synbiotics Corporation, Kansas City, MO 64163 USA

U.S. Vet. License No. 312

1-800-228-4305

www.synbiotics.com

Licensed under U.S. Pat. No. 4,839,275; Canadian Pat. No. 1,232,849; and Australian Pat. No. 582,129.

03-0075-0808

Nac No.

11150485