How Is Hcy Tested?
Patient serum or plasma samples are used in tHcy testing. In the test, serum or plasma is first treated with a reducing agent that converts all Hcy species into the reduced form which is measured either directly or after derivatization. Currently, there are three main tHcy test methods available to the clinical laboratories. They include:
- Chromatographic method
- Immunoassay method
- Enzyme cycling method
The Chromatographic method or HPLC methodbased tHcy test was developed in the early 1980s, and is mainly used in research laboratories.
The Immunoassay-based tHcy test was developed in the mid 1990s, and has been automated for special immunoassay instruments.
The latest technology in tHcy testing is the enzyme cycling-based method that has been developed in the last few years. The enzyme cycling method can be used on any automated clinical chemistry analyzer, and is quickly becoming the preferred method for clinical laboratories.
How do these methods work?
1. Chromatographic Method:
The Chromatographic assay usually uses an HPLC or amino acid analyzer and an ion exchange column to separate derivatized Hcy molecules based on retention times. The quantification is achieved by comparing the Hcy peak area with the peak area of an Hcy standard that is eluted at the same retention time. Each HPLC test takes 10-30 min after sample pre-treatment (another 30-60 min). HPLC testing can only be run sequentially, and is not suited for testing large numbers of samples. It often requires a skilled staff and is a labor-intensive, low-throughput test method. A typical chromatography is shown in the Figure 17.
2. Immunoassay Method:
The immunoassay developed by Axis-Shield is based on the specific binding of an antibody towards the homocysteine enzyme conversion product, SAH. It is a competition assay for binding between SAH from the serum sample and from a tracer that is tagged with a fluorescent chromophore. The detection is based on the changes in fluorescent polarization of the tracer after binding to the antibody (Figure 18). The quantification is achieved through construction of a standard curve with multiple known concentrations of Hcy calibrators.
3. Enzyme Cycling Method:
The enzyme cycling-based tHcy test is the latest method and utilizes the enzyme cycling technique to amplify the detection signal which improves the assay sensitivity. In the assay, protein bound Hcy or oxidized Hcy are first reduced to free Hcy. Next, free Hcy is converted to methionine by the enzyme Hcy methyltransferase using SAM as the methyl donor or co-substrate. The transmethylation reaction converts the co-substrate SAM to SAH (co-substrate conversion product) which is then immediately hydrolzyed into Hcy and Adenosine (Ado) by an enzyme SAH hydrolase. The Hcy thus generated from SAM enters Figure 18 22 into the Hcy conversion reaction catalyzed by Hcy methyltransferase to form a Hcy conversion cycle with a concomitant accumulation of Ado which is detected through a NAD/NADH coupled enzyme reaction at 340 nm. The enzyme cycling Hcy assay scheme is depicted in Figure 19.
The enzymatic tHcy test is a homogenous testing system consisting of 2 or 3 liquid stable reagents. The test is fully automated and is as user friendly as the conventional ALT and AST tests. The test can be used on all automated chemistry analyzers including Beckman CX, LX, Olympus AU series, Hitachi 717 and 917, Cobas Mira, Dade Dimension AR, Bayer Adivia 1650, Abbott Aeroset, etc. The enzyme cycling tHcy test uses a serum or plasma sample of less than 20 μL and the entire test procedure takes only 10 min for either the 2-reagent or 3-reagent system instruments. The test procedure is depicted in Figure 20.
What are the advantages and disadvantages of these tHcy test methods?
There are significant differences among these tHcy testing methods including precision, speed, and cost. Figure 21 depicts these differences.
The HPLC method gives better precision over the immunoassay method, but requires manual sample pre-treatment steps, and therefore is more laborintensive. This method is not widely used in major clinical laboratories.
The Immunoassay method, although automated, requires special instruments that can handle more than 4 reagents per test and a special detection system such as fluorescent polarization (FP). Since the assay involves 4 or more reagents, and multiple steps, it is more expensive, and takes 30-45 min per test. The precision of the immunoassay is often lower than that of HPLC or enzymatic assays.
The enzyme cycling test uses less reagents and is faster on a per-test basis, and therefore is less expensive. It does not require sample pre-treatment and special instruments and can be used on any major automated clinical chemistry analyzers. It is the preferred method, especially for those laboratories routinely testing large numbers of samples.