4.16: Liver Disease, Alkaline Phosphatase

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A liver profile (albumin, total bilirubin, the transaminases, and alkaline phosphatase) and an ammonia assay are performed on a random access analyzer on an aliquot poured off from a phlebotomy tube. The alkaline phosphatase results are zero or negative. Thinking that perhaps the sample has a highly elevated enzyme activity, and the result could be an artifact caused by substrate exhaustion, the technologist repeats the analysis on a tenfold dilution but again obtains very low results; 0 to 5 U/L. The sample is brought to special chemistry for an alkaline phosphatase isoenzyme analysis, and a typical alkaline phosphatase electrophoretic isoenzyme pattern is obtained. The isoenzyme analysis results are inconsistent with the results obtained on the previous analysis for the total alkaline phosphatase activity.

QUESTION

What can the technologist do to verify and possibly explain these results?
Do these additional observations help resolve the problem?

Questions to Consider

What are the reaction conditions for determination of total alkaline phosphatase activity?
What are the principles of the electrophoretic measurement of alkaline phosphatase isoenzymes?
What are the differences between the methods described in question 1 and 2?
Are there any special sample phlebotomy or sample storage conditions required when assaying for alkaline phosphatase?
What anticoagulants can be used to collect whole blood for ammonia analysis?
How does an increased temperature affect the activity of the alkaline phosphatase reaction?

Answer

After considering the above questions, the technologist determines that the blood was not clotted in the phlebotomy tube. Upon checking in the laboratory information system for previous results for this patient, the ‘technologist determines that previous results for alkaline phosphatase ranged from 120 mU/ml to 155 mU/ml. The phiebotomist reports that the sample was kept on ice when delivered to the laboratory. In addition, the technologist performing analysis for the total alkaline phosphatase activity notes that the temperature of the analyzer was two degrees too high.
The technologist should verify the results by going back to the initial phlebotomy tube and determining the patient’s identification. The technologist should check in the laboratory’s information system (computer) for previous alkaline phosphatase results on the patient. Reanalysis on another aliquot from the phlebotomy tube would also be appropriate.
Yes. These observations suggest that the sample was a plasma specimen drawn for the ammonia determination. The most likely explanation for the results, therefore, is that the phlebotomy tube contained EDTA or oxalate as the anticoagulant. EDTA and oxalate are strong chelators and would effectively sequester magnesium ions, inhibiting the alkaline phosphatase reaction. Electrophoresis of the sample would separate the chelator from the isoenzymes and allow the activity to be expressed. The use of the proper phlebotomy tube for ammonia (that is, heparin, see #5 above) would have avoided the problem.

The inappropriately elevated temperature of the analyzer had no bearing on the inhibition of the alkaline phosphatase activity; if anything, it would tend to increase the enzymatic activity.

Answers to Questions to Consider

Alkaline phosphatase has a pH optimum of about 9.5-10.5 and has an absolute requirement for Mg++ as a cofactor (See Method Alkaline Phosphatase on CD-ROM). Many molecules can act as a phosphate donor (substrate), although para-nitrophenyl phosphate is the most frequently used substrate (See Method Alkaline Phosphatase on CD-ROM).
Alkaline phosphatase isoenzymes carry different net charges and can be separated by polyacrylamide gel electrophoresis (see p 206-207) and identified as discrete bands of enzyme activity (See Method Alkaline Phosphatase on CD-ROM). The enzyme activity of the separated fractions is determined by addition of substrate reagent to the gels.
The electrophoretic method physically separates the isoenzymes from each other and from the serum matrix on the basis of charge differences. The method for measuring total akaline phosphatase activity is a kinetic enzymatic assay performed on the diluted serum matrix. The primary difference between the methods is the presence of the sample matrix in the total enzyme activity assay.
A red top serum phlebotomy tube is preferred, although the analysis can be performed on heparinized plasma. The isoenzymes are stable for a week at 4°C and longer when frozen (See Method Alkaline Phosphatase on CD-ROM).
Plasma samples for ammonia can use heparin, citrate, EDTA or oxalate in the collection tubes. The preferred anticoagulant is sodium heparin.
An increase in temperature of 2° over the standard temperature would serve to increase enzymatic activity (See Chapter 54). This temperature difference is not great enough to heat denature the enzyme.