ACID PHOSPHATASE PROCEDURE

Intended for the Quantitative Determination of Prostatic Acid Phosphatase in Serum

SUMMARY AND EXPLANATION

"Acid Phosphatase" is the term used to describe all phosphatase activity with optima below pH = 7.0. Although significant levels of this enzyme activity appear in several tissues, the prostatic acid phosphatase has the greatest clinical interest since high elevations of this isoenzyme occur in sera of patients who have prostatic carcinoma with metastases. Following therapy, the serum levels gradually approach normal. If the malignancy is localized, serum acid phosphatase usually remains normal or slightly elevated (1).

Roy, et al (2) recommended sodium thymolphthalein monophosphate as a substrate for prostatic acid phosphatase because of the high degree of selectivity shown in the reaction. Clinical evaluation of this method has confirmed its superiority for use in the differential diagnosis of prostatic carcinoma (3).

The EAGLE ACID PHOSPHATASE PROCEDURE employs a modification of Roys method proposed by Ewen and Spitzer (4). The modification includes changes in buffer type, pH and substrate concentration. Under these conditions, prostatic acid phosphatase has markedly increased reactivity.

PRINCIPLE

Prostatic acid phosphatase hydrolyzes sodium thymolphthalein monophosphate releasing thymolphthalein. The addition of alkali terminates the enzymatic reaction and simultaneously develops a blue color. The intensity of blue color measured at 590 nm is proportional to enzyme activity.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2000 provides:

ACID PHOSPHATASE REAGENT CONCENTRATE - (Cat. No. 2001)

REACTIVE INGREDIENTS:

2.6 nM sodium thymolphthalein monophosphate. Buffer and stabilizer added.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 2 - 8° C. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, orange solution. Turbidity would indicate deterioration.

ACID PHOSPHATASE ACID DILUENT - (Cat. No. 2002)

REACTIVE INGREDIENTS:

50 mM acetic acid.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15 - 30° C. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, colorless solution. Turbidity would indicate deterioration.

PHOSPHATASE COLOR DEVELOPER - (Cat. No. 2003)

REACTIVE INGREDIENTS:

100 mM Sodium Hydroxide and 100 mM Sodium Carbonate.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15 - 30° C. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, colorless solution. Turbidity would indicate deterioration.

ACID PHOSPHATASE CALIBRATOR - (Cat. No. 2004)

REACTIVE INGREDIENTS:

0.3 mM thymolphthalein. Stabilizer added. Equal to 10 U/L enzyme activity in the method.

PRECAUTIONS:

Contains alcohol. Do not inhale or ingest.

STORAGE AND STABILITY:

Store at 15 - 30° C. Stable until expiration date if sealed tightly.

DETERIORATION:

The calibrator should be a clear, colorless solution. Cloudiness would indicate deterioration.

ACID PHOSPHATASE PRESERVATIVE - (Cat. No. 2005)

REACTIVE INGREDIENTS:

5.0 M acetate buffer, pH = 5.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15 - 30° C. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, colorless solution. Turbidity would indicate deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 590 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1.Unhemolyzed serum is preferred.

2.Plasma (EDTA) may be used.

3. Jaundiced or lipemic samples require a Serum Blank.

4. Separate the serum or plasma from the erythrocytes within two hours of collection.

SAMPLE STORAGE:

Acid phosphatase in serum and plasma is labile at room temperature. The sample should be acidified by adding 0.02 mL ACID PHOSPHATASE PRESERVATIVE (Cat. No. 2005) to each mL of sample. After acidification, the sample is stable several hours at room temperature or for up to a week stored at 2 - 8° C (1).

ADDITIVES:

Acidify serum with preservative as instructed in SAMPLE STORAGE.

INTERFERING SUBSTANCES:

1. Flouride, oxalate and heparin inhibit acid phosphatase and should not be used to anicoagulate blood specimen.

2. Young et al (5) have reviewed drug effects on serum acid phosphatase.

PROCEDURE

MATERIALS PROVIDED:

ACID PHOSPHATASE REAGENT CONCENTRATE (Cat. No. 2001, ACID PHOSPHATASE ACID DILUENT (Cat. No. 2002) and PHOSPHATASE COLOR DEVELOPER (Cat. No. 2003).

AUTOMATED PROCEDURE:

The method is not applicable to automated instruments.

PREPARATION OF WORKING REAGENT:

As an alternative to STEPS 1 and 2 in MANUAL PROCEDURE, a single Working Reagent can be prepared. Add the entire contents of the ACID PHOSPHATASE ACID DILUENT to the entire contents of the ACID PHOSPHATASE REAGENT CONCENTRATE and mix well. WHEN STORED IN THE AMBER BOTTLE at 2-8° C, THE WORKING REAGENT CAN BE USED UNTIL THE EXPIRATION DATE. The Working Reagent should be clear and colorless with pH = 5.3 - 5.5. Turbidity would indicate deterioration. If, after addition of PHOSPHATASE COLOR DEVELOPER to the Reagent Blank, its absorbance measured against water exceeds 0.500, the Working Reagent should not be used.

MANUAL PROCEDURE:

1. Dispense 0.5 mL of ACID PHOSPHATASE Reagent Concentrate into tubes labeled: Reagent Blank, Calibrator, Control, Sample 1, etc.

2. Dispense 0.5 mL OF ACID PHOSPHATASE ACID DILUENT into each tube and mix well. NOTE: As an alternative to STEPS 1 and 2, dispense 1.0 mL of the Working Reagent into each tube.

3. Pre-warm the tubes to 37º C (approximately 5 minutes).

4. At timed intervals, add 0.1 mL of each sample to its respective tube and mix gently. Use deionized water as sample for Reagent Blank.

5. Incubate for exactly 30 minutes at 37º C.

6. Following the same sequence as in STEP 4, add 1.0 mL PHOSPHATASE COLOR DEVELOPER at timed intervals and mix well.

7. Adjust instrument to zero absorbance at 590 nm using the Reagent Blank.

8. Read and record absorbance values for Calibrator, Control and Unknowns.

NOTE: For a direct read-out instrument, set read-out to concentration value of Calibrator and read the Unknown concentrations directly.

PROCEDURAL NOTE:

Linearity extends to 100 U/L for the method. However, the absorbance for enzyme activity above 20 U/L may exceed the instruments limitation. In this case, dilute the test solution and Reagent Blank from STEP 6 five-fold (X 5) with PHOSPHATASE COLOR DEVELOPER. Repeat the absorbance reading for the diluted test after zeroing the instrument with the diluted Reagent Blank. Multiply the absorbance reading by the dilution factor (X 5) when calculating the Unknown. If the absorbance of the diluted test solution exceeds the instruments limitation, then the enzyme activity is greater than 100 U/L. In this case, dilute the sample ten-fold (X 10) with 0.9% sodium chloride solution and repeat the assay. Multiply the answer by the dilution factor (X 10).

IMPORTANT NOTE:

Usually a Serum Blank is not needed for this procedure. However, it is recommended that any patient sample having a value between 2.0 - 3.0 U/L be blanked to determine if the enzyme activity is slightly elevated or high normal. Jaundiced and lipemic samples require a Serum Blank.

SERUM BLANK:

1. Add 0.5 mL ACID PHOSHATASE REAGENT CONCENTRATE and 0.5 mL of ACID PHOSPHATASE ACID DILUENT to a tube. NOTE: Alternatively, 1.0 of Working Reagent may be used.

2. Add 1.0 mL PHOSPHATASE COLOR DEVELOPER. Mix well.

3. Add 0.1 mL of sample and mix gently.

4. For jaundiced and lipemic samples, incubate 30 minutes at 37º C. Clear specimens do not require an incubation.

5. Zero instrument at 590 nm with Reagent Blank.

6. Read and record absorbance of Serum Blank.

7. Subtract blank absorbance from test absorbance measured in STEP 8 of MANUAL PROCEDURE. Use this value in calculating acid phosphatase activity.

STABILITY OF FINAL REACTION:

The test samples should be read within 60 minutes of color development.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since it is linear in range of 0 - 100 U/L. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. ACID PHOSPATASE CALIBRATOR (Cat. No. 2004) is supplied for this purpose.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and elevated) analyzed in the same manner used for the Unknowns. Lypolized control sera should be acidified when reconstituted (see SAMPLE STORAGE). EAGLE DIAGNOSTICS offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No.8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

CALCULATION OF RESULTS

Use the following equation to determine Unknown concentrations:

Unknown (U/L) =

Unk. Abs.

-------------  X Cal. Conc. (U/L)

Cal. Abs.

EXAMPLE:

A 10 U/L Calibrator head Abs. = 0.580 while the Unknown Abs. = 0.200. The Alkaline Phosphatase activity of the Unknown is:

0.200

------     x 100 U/L     =     3.4 U/L

0.580

LIMITATIONS

1. Incubation temperature should be controlled to + 0.1° C.

2. Because the reaction measures prostatic acid phosphatase directly, along with some platelet acid phosphatase, results will differ from those of indirect methods employing tartrate-inhibition. Sodium thymolphthalein monophosphate is hydrolyzed by erythrocytic acid phosphatase at a very slow rate.

3. Patients with lymphoproliferative disorders and markedly elevated platelet counts may show an acid phosphatase activity more than twice the upper normal limit. The platelet acid phosphatase can be differentiated from prostatic acid phosphatase in such patients by comparing the activity in a platelet-free EDTA plasma with that of serum. If the elevated activity in serum is of platelet origin, the activity falls within the normal range in platelet-free plasma (4).

EXPECTED VALUES                                                      0.5 - 2.0 U/L

This range represents the 95% confidence obtained from a clinically normal population (1). No apparent difference exists between male and female specimens indicating that acid phosphatase in normal sera appears to be of non-prostatic origin (4, 6). Elevations of enzyme activity may reach 40 - 50 times the upper limit of normal in males with prostatic cancer with metastases. However, when the carcinoma remains localized in the prostate gland, normal or only slightly raised levels of enzyme activity is found. After surgery or estrogen therapy, the levels slowly approach normal, with a subsequent rise if treatment is unsuccessful (1).

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 100 U/L.

PRECISION:

Within Run - Normal and abnormal control sera were assayed 20 times each to establish within run precision.

MEAN / STD. DEV. / %CV

Normal 1.43 / 0.03 / 2.1

Abnormal 6.70 / 0.07 / 1.1

Run to Run - Normal and abnormal control sera were assayed for 10 working days to establish run to run precision.

Abnormal 6.50 / 0.15 / 2.3

SPECIFICITY:

Sodium thymolphthalein monophosphate demonstrates a high degree of specificity for the prostatic acid phosphatase and reacts to a much lesser extent with the platelet and erythrocytic acid phosphatases (7). This ACID PHOSPHATASE PROCEDURE showed a 99% correlation when compared with another widely used commercial method that also employs sodium thymolphthalein monophosphate as a substrate.

SENSITIVITY:

his ACID PHOSPHATASE PROCEDURE has a sensitivity of 0.02 U/L per 0.001 absorbance unit.

REFERENCES

1. Saunders Co., Philadelphia, 1986, p. 752.

2. Roy, A.V., Brower, M.E., and Hayden, J.E., Clin. Chem. 17, 1093 (1971).

3. Ladenson, J.H., and McDonald, J.M., Clin. Chem. 24, 129 (1978).

4. Ewen, L.M., and Spitzer, R.W., Clin. Chem. 22, 627 (1976).

5. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. 21, 241D (1975).

6. Li, C.Y., Chuda, R.A., Lam, W.K.W., and Yam, L.T., J. Lab. Clin. Med. 82, 446 (1973).

7. Foti, A.G., Herschman, H., and Cooper, J.F., Clin. Chem. 23, 95 (1977).