High-density lipoproteins (HDL) is a type of lipoprotein that carries cholesterol from the peripheral tissues to the liver. In the liver excretion of cholesterol takes place. Thus, HDL-cholesterol is considered good cholesterol. That means greater is the value of HDL reduces the chances of heart diseases.
Everything that I state below will be based on a semi-auto biochemistry analyzer and can differ from one manufacturer to another.
When the serum reacted with the precipitation reagent and then centrifuged, the HDL will remain as a supernatant. Low-density lipoproteins( LDL), and very-low-density lipoproteins (VLDL) will precipitate out.
After that, HDL is assayed with the help of the cholesterol (CHOD/PAP) reagent.
Normally, you can store the reagents at 2-80C till the expiration date. But, once you open the bottle, the lifespan of reagents and the working solution will reduce to about 2 months (at 2-80C).
- Wear an apron and surgical gloves before carrying out the measurement.
- Look for the expiry date of the reagents during purchase and measurement time. Suppliers tend to give you reagent kits with a low expiry interval.
- Bring the reagents and samples to room temperature before you can carry out any measurement.
- Always store reagents in the refrigerator when not in use.
Mix 100 μl of serum to the 100 μl of precipitating reagent. Then incubate the mixture at room temperature for about 5 mins. Finally, centrifuge it at 4000 rpm for 2 mins to obtain the clear supernatant. An unclear supernatant can be an indication of the incomplete precipitation of lipoproteins or part of the precipitate is floating on the surface. In both cases, there will be a high concentration of triglycerides (TG). A high TG value (above 1000 mg/dl) will give you an error in your measurement. In that case, again dilute the precipitate solution with distilled water or 0.9% NaCl at a ratio of 1: y. Repeat the steps from the beginning. Finally, when you obtain the value of HDL-CHOL, multiply it with the dilution factor (1+y).
If you have two reagents, then you can make a working solution out of it. Go, through your literature to know the composition. Then add 50 μl or 100 μl (go through your literature) of standard or supernatant to 100 μl of the prepared working solution (or a single reagent). Then you can incubate the solution in water or dry bath at 370C for about 5 mins. After that, feed the solution into a semi-auto biochemistry analyzer and get the result.
In case of a reagent blank, you can feed 1000 μl of working solution or a single reagent(if you have got only one reagent besides standard reagent) directly into the machine. However, I have seen the operators feeding distilled water for both water and reagent blank.
You will get, an increasing curve in the biochemistry analyzer.