Check4® Urine Myoglobin

Check4® Urine Myoglobin is a rapid qualitative test that can detect human myoglobin in diluted urine. The method employs a unique combination of monoclonal-dye conjugate and polyclonal solid phase antibodies to identify myoglobin in the test samples with a high degree of specificity.

As the sample flows through the absorbent device, the labelled antibody-dye conjugate binds to the myoglobin forming an antibody antigen complex. This complex binds to the anti-myoglobin antibody in the positive reaction zone (B) and produces a pink-rose colour band when myoglobin concentration is higher than 100 ng/ml. In the absence of myoglobin, there is no line in the positive reaction zone.

The reaction mixture continues flowing through the absorbent device past the positive reaction zone and control zone. Unbound conjugate binds to the reagents in the control zone (C) producing a pink-rose colour band, demonstrating that the reagents are functioning correctly.

Myoglobin is an intra cellular haem protein involved in the storage and transfer of oxygen to muscle tissues. Its presence outside of the muscle cells is an indicator of muscle damage by physical or chemical agents, or restriction of oxygen or energy supply to the muscle. When released in this way, myoglobinuria results. This is the presence of myoglobin in the urine, resulting in a brown colour when present at high concentration.

Urine myoglobin can cause acute renal failure by precipitation in the renal tubules and by conversion to products toxic to the tubules. Rapid diagnosis and treatment is therefore needed if recovery is to follow. Although muscle injury leading to myoglobinuria can be caused in a number of ways (e.g. severe electric shock, drug overdose and excessive exercise) crush injury is a common cause.

1. This test is designed for Research Use Only (RUO).
2. Please read the instruction leaflet carefully before using this test.
3. Do not use beyond the expiry date which appears on the package label.
4. Do not use a test from a damaged protective wrapper.
5. All reagents and materials coming in contact with potentially infectious specimens must be treated with appropriate disinfectants or autoclaved at +121°C for at least one hour.
6. Wear protective clothing such as laboratory coats and disposable gloves while assaying samples.
7. Avoid any contact between hands and eyes or nose during specimen collection and testing. Extraction reagents may cause irritation to skin, eyes and mucus membranes. Wash off immediately if extraction reagents come into contact with skin.
8. Do not eat or drink in the area where specimens and kit reagents are handled.

1. As it is the case with any diagnostic procedure, the physician should confirm the data obtained using this test by other clinical methods.
2. Check4® Urine Myoglobin is designed to yield a positive result for myoglobin concentration at 100 ng/ml or higher. As no commercially available standard exists Check4® Urine Myoglobin is quality controlled against bio-engineered protein obtained from human heart. The range of tested concentrations is 0-400ng/ml. No hook effect is seen at 400ng/ml, however urine concentrations can exhibit extremely high levels which may lead to a hook effect unless the dilution regime for negatives is adhered to.
3. Check4® Urine Myoglobin only provides qualitative results.
4. Use only fresh urine specimens.
5. The diluent contains buffer however pH is important for sample stability and samples should be buffered as section V-sample collection.
6. Some drugs such as diphenhydramine hydrochlorate have an effect on renal function and therefore could modify urine clearance. It is possible that this could cause false positive results.

If there is no distinct coloured band visible in the control window, the test is invalid. It is recommended that in this case the test be repeated with a fresh device.

Results should be read after ten minutes.

A. Sensitivity
The analytical sensitivity of the test is 100 ng Myoglobin/ml. Concentrations below 100 ng/ml may also be detected occasionally.

B. Specificity

An evaluation of Check4® Urine Myoglobin for the detection of urine myoglobin was carried out at a major UK teaching hospitals which receives a significant number of motor accident victims.

Initially the test sensitivity caused issues: The detection level of Check4® Urine Myoglobin is 100ng myoglobin/ml. Since concentrations of 300ng to 2g myoglobin/ml may be found in myoglobinuria it was clear that prozoning could occur (i.e. the appearance of false negative results due to overloading of the capture antibody). To avoid this, dilutions were made before testing with Check4® Urine Myoglobin.

The evaluation was made with 64 urine samples taken from crush injury victims. Two diagnostic methods were used on each sample, a dipstick-based method in which haemoglobin and myoglobin were separated by centrifugal filtration before the test and the modified Check4® Urine Myoglobin protocol. Samples were scored as either positive or negative for myoglobin by each method.

The results in all but one sample agreed for both methods. This sample was further diluted and it then gave a myoglobin positive result in agreement with the dipstick test.

A false negative seen with an epileptic patient has resulted in a modification of the Check4® Urine Myoglobin protocol to increase the secondary dilution in order to confirm true negatives.