What specimen preparation is commonly used to perform alkaline phosphatase isoenzyme determinations?

What specimen preparation is commonly used to perform alkaline phosphatase isoenzyme determinations?

• A. Serum is divided into 2 aliquots, one is frozen and the other is refrigerated
• B. Serum is divided into 2 aliquots, one is heated at 56 C and the other is unheated
• C. No preparation is necessary since the assay uses EDTA plasma
• D. Protein-free filtrate is prepared first

Answer: (B)

Isoenzyme determinations for alkaline phosphatase rely on differences in heat stability among the isoforms. Bone alkaline phosphatase is relatively heat-stable, whereas the hepatic and intestinal forms are more heat-labile. By splitting serum into two portions and heating one to 56°C for about 10 minutes, the heat-labile isoenzymes are inactivated while the bone isoenzyme remains active. The unheated portion provides total ALP activity, so comparing the heated and unheated results lets you estimate the proportions of bone versus non-bone isoenzymes. This approach uses serum, not EDTA plasma, because anticoagulants and the heating step can affect enzyme activity. Other preparative ideas, like freezing, using a protein-free filtrate, or heating only one aliquot without a direct comparison, do not achieve the needed differential separation of isoforms.

Isoenzyme determinations for alkaline phosphatase rely on differences in heat stability among the isoforms. Bone alkaline phosphatase is relatively heat-stable, whereas the hepatic and intestinal forms are more heat-labile. By splitting serum into two portions and heating one to 56°C for about 10 minutes, the heat-labile isoenzymes are inactivated while the bone isoenzyme remains active. The unheated portion provides total ALP activity, so comparing the heated and unheated results lets you estimate the proportions of bone versus non-bone isoenzymes. This approach uses serum, not EDTA plasma, because anticoagulants and the heating step can affect enzyme activity. Other preparative ideas, like freezing, using a protein-free filtrate, or heating only one aliquot without a direct comparison, do not achieve the needed differential separation of isoforms.