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ACID PHOSPHATASE (KINETIC) PROCEDURE
Intended for the Quantitative Determination of Acid Phosphatase in Serum
SUMMARY AND EXPLANATION
"Acid Phosphatase" is the term used to describe all phosphatase activity with optima pH
below 7.0. Although significant levels of this enzyme activity appear in several tissues,
the prostatic acid phosphatase has the greatest clinical interest since high levels 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 to slightly elevated (1).
Many phosphate compounds such as phenylphosphate, a-glycerolphosphate, p-nitrophenylphosphate and thymolphthalein phosphate have been used as substrates in the
measurement of acid phosphatase. Most of these have been found to lack sensitivity or be
too sensitive to the non-prostatic fraction. In 1971, Roy, et al (2) proposed a method
using sodium thymolphthalein monophosphate as a substrate. This method was later
modified by Ewen and Spitzer (3) and was shown to offer a high degree of selectivity for
the prostatic fraction and became an excellent manual method for prostatic acid
phosphatase determinations, however, it was limited in the fact that the TMP method
could not be easily adapted to automated instrumentation. In 1959, Babson (4) had
proposed a method using Alpha-naphthylphosphate as a substrate but was found not to be
specific for the prostatic fraction. Later, in 1971, Hillman (5) modified the Babson
method to include a diazotized 2-amino-5-chlorotoluene (Fast Red TR) to form a diazo
dye that would absorb strongly at 405 nm. L-tartrate was used as a specific inhibitor of
prostatic acid phosphatase to establish a way to differentiate the prostatic from the non-prostatic isoenzyme (6).
The EAGLE ACID PHOSPHATASE (KINETIC) PROCEDURE employs these
modifications to offer a kinetic method that is specific, fast, simple and which can easily
be adapted to automated instrumentation.
PRINCIPLE
Serum acid phosphatase hydrolyzes a-napthylphosphate to a-naphthol and inorganic
phosphate. The a-naphthol produced is instantly coupled with Fast Red TR to form a
colored complex which can be measured at 405 nm. The rate of hydrolysis is
proportional to the enzyme activity present. L-tartrate is used to inhibit the prostatic
fraction while not interfering with the reaction sequence. Therefore, testing is performed
with and without the presence of L-tartrate so that the difference between the two assays
yields the level of prostatic acid phosphase present in the sample.
REAGENTS: FOR IN-VITRO DIAGNOSTIC USE
Reagent Set Cat. No. 2010 provides:
ACID PHOSPHATASE (KINETIC) REAGENT - (Cat. No. 2011)
REACTIVE INGREDIENTS:
After reconstitution, 3.0 mM a-naphthylphosphate, 1.0 mM Fast Red TR. 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 - 8º C. Unopened vials are stable until expiration date if sealed tightly.
Reconstitute vials with volume of D-H2O stated on vial label. Swirl gently to dissolve.
After reconstitution, the reagent is stable for 24 hours when stored at controlled room
temperature (22 - 28º C) or for 14 days when stored refrigerated (2 - 8º C).
DETERIORATION:
The reagent should be a clear, colorless solution. Do not use if the reconstituted reagent
has an absorbance of 0.300 when measured against water at 405 nm.
L-TARTRATE REAGENT - (Cat. No. 2012)
REACTIVE INGREDIENTS:
After reconstitution, 2.0 mM sodium L-tartrate in a buffer solution.
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 - 8º C. Unopened vials stable until expiration date if sealed tightly.
Reconstitute with volume of D-H2O stated on vial label. Warm reagent gently to 40 - 50º
C to aid in dissolution if needed. After reconstitution, the reagent is stable until
expiration date on the vial label stored at 2 - 8º C. Crystallization may occur during
refrigerated storage; if crystals are present, warm the reagent to 40 - 50º C until
dissolved. This may be repeated as often as needed without effecting stability.
DETERIORATION:
Precipitation which will not redissolve after applying heat would indicate deterioration of
this reagent and the reagent should not be used.
ACID PHOSPHATASE PRESERVATIVE - (Cat. No. 2005)
REACTIVE INGREDIENTS:
5.0 mM 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 405 nm.
SPECIMEN COLLECTION
PRECAUTIONS:
1. Use unhemolyzed serum .
2. Separate the serum from the clot within two hours of collection.
SAMPLE STORAGE:
Acid phosphatase in serum is extremely 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).
INTERFERING SUBSTANCES:
1. Flouride, oxalate and heparin inhibit acid phosphatase activity and should not be used to anticoagulate blood specimen.
2. Young et al (7) have reviewed drug effects on serum acid phosphatase.
PROCEDURE
MATERIALS PROVIDED:
ACID PHOSPHATASE (KINETIC) REAGENT (Cat. No. 2011), L-TARTRATE
REAGENT (Cat. No. 2012), and ACID PHOSPHATASE PRESERVATIVE (Cat. No.
2005).
MATERIALS REQUIRED BUT NOT PROVIDED:
1. 0.1 mL and 0.01 micropipettors
2. 1.0 mL pipettor or dispenser
3. Incubator capable of maintaining 37° C
4. Timer, test tubes and rack
5. Instrument capable of reading at 405 nm with a temperature control
cuvette well that can maintain 37° C
REACTION CONDITIONS:
Wavelength 405 nm
Reaction Type Kinetic
Preincubation Time 5 minutes
Reaction Time 1 minute
Reaction Temperature 37° C
Sample Volume 0.1 mL
Reagent Volume 1.0 mL
L-Tartrate Volume 0.01 mL
Total Volume 1.11 mL
Low Normal 0.0 U/L
High Total Value 11.7 IU/L
High Prostatic Value 3.5 IU/L
Factor (Total) 853
Factor (Prostatic) 860
PREPARATION OF WORKING REAGENT:
Reconstitute the reagents with the amount of D-H2O indicated on the vial label. Swirl to
dissolve. DO NOT SHAKE. The L-Tartrate reagent may require warming to 40 - 50° C to
effect dissolution.
AUTOMATED PROCEDURE:
Refer to specific instrument application for instructions.
MANUAL PROCEDURE:
PROCEDURAL NOTES:
1. For instruments requiring a total volume 1.0 mL, increase ACID PHOSPHATASE (KINETIC) REAGENT volume to 2.0 mL and volume of samples to 0.2 mL and L-TARTRATE volume to 0.02 mL and proceed as outlined.
2. Samples with values 35 IU/L should be diluted with 0.9% sodium chloride
solution and reassayed. Multiply the test result by the dilution factor to obtain the final
answer.
A. TOTAL ACID PHOSPHATASE DETERMINATION
1. Dispense 1.0 mL of ACID PHOSPHATASE (KINETIC) REAGENT into tubes labeled: Control, Sample 1, etc.
2. Place 0.1 mL of Specimen into appropriately labeled tube. Mix well.
3. Incubate at 37° C for 5 minutes.
4. Zero instrument at 405 nm using D-H2O.
5. Read and record absorbance exactly every minute for 5 minutes to determine ^A / minute.
6. Obtain values by multiplying the ^A / minute X 853. (See Calculation of Results)
B. NON-PROSTATIC ACID PHOSPHATASE DETERMINATION
1. Dispense 1.0 mL of ACID PHOSPHATASE (KINETIC) REAGENT into tubes labeled: Control, Sample 1, etc. and mix.
3. Place 0.1 mL Specimen into appropriately labeled tube. Mix well.
4. Incubate at 37° C for 5 minutes.
5. Zero instrument at 405 nm using D-H2O.
6. Read and record absorbance exactly every minute for 5
7. Obtain values by multiplying the ^A / minute X 860. (See Calculation of Results)
C. PROSTATIC ACID PHOSPHATASE DETERMINATION
This value is obtained by subtracting the result of the non-prostatic assay (B) from the
total acid phosphatase assay (A).
STABILITY OF FINAL REACTION:
This is a kinetic reaction. The timings must be accurate as the reaction never reaches an
end-point.
CALIBRATION:
The reaction is standardized by means of the molar absorptivity of a-naphthol Fast Red
TR complex at 405 nm.
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.
Lypholized 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
One International Unit (IU/L) is defined as the amount of enzyme that catalyzes the
transformation of one micromole of substrate per minute. The calculation factors of
Total = 853 and Non-Prostatic = 860 were derived from the following equations:
Where:
(A1 - A2) = Change in Absorbance
1.10 = Total reaction volume of Total
1.11 = Total reaction volume of Non-Prostatic
12.9 = Millimolar absorptivity of a-naphthol Fast Red complex at 405 nm
1.0 = Light path in cm
0.1 = Specimen volume in mL
A. Total Acid Phosphatase Calculation:
IU/L =
(A1 - A2) X 1.10 X 1000
--------------------- or (A1 - A2) X 853 = IU/L
1.0 X 12.9 X 0.1
B. Non-Prostatic Acid Phosphatase Calculation:
IU/L =
(A1 - A2) X 1.11 X 1000
---------------------- or (A1 - A2) X 860 = IU/L
1.0 X 12.9 X 0.1
C. Prostatic Acid Phosphatase Calculation:
Total Fraction (A) - Non-Prostatic Fraction (B) = Prostatic
EXAMPLE:
Total Acid Phosphatase reaction had a change in absorbance (A1 - A2) of 0.010 X 853
(factor) = 8.5 IU/L, while the Non-Prostatic reaction had a change in absorbance of 0.009
X 860 (factor) = 7.7 IU/L. The Prostatic Acid Phosphatase value = 0.8 IU/L (8.5 - 7.7).
LIMITATIONS
Incubation temperature should be controlled to + 0.1° C.
EXPECTED VALUES (8)
Total Acid Phosphatase: 0.3 - 11.7 IU/L
Prostatic Acid Phosphase: 0.0 - 3.5 IU/L
This range represents the 95% confidence interval obtained from a clinically normal
population. Each laboratory should establish its own range of expected values.
PERFORMANCE CHARACTERISTICS
LINEARITY:
This method is linear to 35 IU/L.
PRECISION:
Within Run - Normal and abnormal control sera were assayed 20 times each to establish
within run precision.
MEAN / STD. DEV. / %CV
Normal 8.7/ 0.14 / 1.6
Abnormal 33.3 / 0.29 / 0.9
Run to Run - Normal and abnormal control sera were assayed for 10 working days to establish run to run precision.
Normal 8.7 / 0.28 / 3.6
Abnormal 32.7 / 0.36 / 1.1
SPECIFICITY:
A comparison of this ACID PHOSPHATASE (KINETIC) PROCEDURE with a
commercial method using a similar reagent system showed a 99.8% correlation with
samples in the normal and abnormal range.
SENSITIVITY:
This ACID PHOSPHATASE (KINETIC) PROCEDURE has a sensitivity of 0.8 U/L per
0.001 absorbance unit.
REFERENCES
1. Chemistry, 2nd. ed., W.B. Saunders Co., Philadelphia, p. 882 (1994).
2. Roy, A.V., Brower, M.E., and Hayden, J.E., Clin. Chem. 17, p. 1093 (1971).
3. Ewen, L.M., and Spitzer, R.W., Clin. Chem. 22, 627 (1976).
4. Babson, A.L., et al, Am. J. Clin Path Vol. 32, p. 83 (1959).
5. Hillman, G.Z., Klin Chem. Klin Biochem., Vol. 3, p. 273 (1971).
6. Fabiny-Byrd, D.L., Ertingshausen, G., Clin. Chem. Vol. 13, p. 841 (1972).
7. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. 21, 241D (1975).
8. Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders Co., Philadelphia, p. 618 (1976).
