1.0 INTENDED
USE
This
reagent is intended for the quantitative determination of Acid Phosphatase (ACP)
in serum.
2.0
BACKGROUND
2.1 METHOD AND
HISTORY
Several
substrates have been used for determination of acid phosphatase activity
(10.3-10.6.) However, a-naphthyl
phosphatase has been recommended as the most suitable substrate for automated
assays (10.7, 10.8.) This method
measures the acid phosphatase activity using a-naphthyl phosphate as
substrate. The reagents may be
employed to determine both the tartrate resistant and tartrate sensitive
fractions of the enzyme.
2.2 TEST
PRINCIPLE
Acid
phosphatase (ACP) catalyzes the hydrolysis of a-naphthyl phosphate to a-naphthol
and inorganic phosphate. a-Naphthol
immediately reacts with fast red TR salt to produce a yellow chromophore with an
absorbance maximum at 405nm. The
rate of increase in absorbance at 405nm is directly proportional to ACP activity
in the sample. ACP activity is
determined in the presence and absence of L-tartrate, the difference in activity
being attributed to prostatic acid phosphatase activity.
ACP
a-Naphthyl Phosphate + H2O ‑‑-‑> a-Naphthol + inorganic
phosphate
a-Naphthol + Fast Red TR ‑‑-‑> Chromophore
2.3 CLINICAL
SIGNIFICANCE
Acid
phosphatase (orthophosphoris phosphohydrolase) is found in a variety of tissues,
notable in the prostate, stomach, liver, muscle, skin, spleen and erythrocytes
(10.1.) However, acid phosphatase
activity in human serum from healthy donors is relatively low compared to the
activity of alkaline phosphatase.
Serum acid phosphatase activity is increased in some prostate cancer
patients, especially the enzyme activity which is tartrate sensitive. Therefore, determination of serum acid
phosphatase activity is of established value in the detection of metastasizing
carcinoma of the prostate (10.2.)
3.0 SPECIMEN COLLECTION AND
HANDLING
3.1 PATIENT
PREPARATION
No special patient preparation is required.
3.2 SPECIMEN
COLLECTION.
Fresh,
clear, unhemolyzed serum is the preferred specimen. Plasma should not be used. Separate serum from clot promptly after
blood collection.
Use a standard venipuncture tube to draw patient
sample.
The
amount of sample required will depend on the analyzer used. The amount of serum required is in the
range of 5-100 µl. Call Biotron's
technical service department at 1-800-595 TRON for the recommended sample volume
for your analyzer.
Record
the patient's name, date and time of sample collection and
preparation.
3.3 SPECIMEN
STORAGE
Acid
phosphatase at the normal pH of serum is very labile. To stabilize the enzyme, add 20µl ACP
Stabilizer to 1ml serum. Mix and
store serum in refrigerator (2-8°C) until ready to assay. Acid phosphatase activity in serum
treated in this manner will remain stable for 7 days.
4.0 MATERIALS
(10
X 10 ml)
(6 X 50
ml)
Reagents necessary for the determination of acid phosphatase are included in the kit.
4.1
REAGENT
4.1.1 ACP Reagent
contains:
a-naphthyl
phosphate
4 mmol/L
Fast
Red TR
1 mmol/L
nonreactive
stabilizers and fillers
4.1.2 ACP Tartrate Reagent
contains:
L-Tartrate
2 mol/L
nonreactive
stabilizers and fillers
4.1.3 ACP Stabilizer
contains:
Acetate
Buffer
5 mol/L
4.2 WARNINGS AND
PRECAUTIONS
For In Vitro Diagnostic Use. Not for Internal use in Humans or Animals. In Vitro Diagnostics reagents may be hazardous. Avoid ingestion and skin or eye contact.
4.3 REAGENT
PREPARATION
Reconstitute
ACP reagent with volume of deionized water indicated on the vial label. Replace rubber stopper and allow to sit
for 5 minutes. Swirl gently until
dissolution is complete.
Reconstitute ACP tartrate reagent with 5 ml of deionized water. Replace rubber stopper and allow to sit
for 5 minutes. Swirl gently until
dissolution is complete.
Record the date and time of reconstitution.
The
ACP Stabilizer is supplied in liquid form and requires no further
preparation.
4.4 REAGENT STORAGE AND
STABILITY
When stored at 2°-8°C, away from light, unopened reagents are stable until the expiration date printed on the label.
Reconstituted
ACP reagent is stable for 2 weeks at 2°-8°C or 24 hours at
18°-26°C.
Reconstituted
ACP Tartrate reagent is stable indefinitely at 2°-8°C if free from bacterial
contamination. If crystallization
occurs, warm reagent (40-50° C) to dissolve.
ACP
reagent is not suitable for use if the absorbance of the reconstituted solution
is greater than 0.5 at 405nm.
Discard vial if reagent exhibits caking due to possible moisture
penetration, does not dissolve completely upon reconstitution or if solution
appears turbid.
ACP
tartrate reagent and ACP stabilizer are not suitable for use if the solution
appears turbid.
4.5 ADDITIONAL MATERIALS
REQUIRED
4.5.1 A spectrophotometer or colorimeter
capable of reading absorbance accurately at 405 nm.
4.5.2 1 cm cuvettes or a flow cell capable of
transmitting light at 405 nm.
4.5.3 Test tubes capable of holding 2
ml.
4.5.4 Pipettes capable of delivering 1 ml and
100 µl.
4.5.5 Timer with one minute
increments.
4.5.6 Constant temperature heat source which
can be adjusted to 30° C or 37° C.
4.5.7 Normal and abnormal control for quality
control.
5.0 TEST
PROCEDURE
The following is a general procedure for use on a manual
instrument.
5.1 PROCEDURE
CONDITIONS
Wavelength
405 nm
Temperature
30° C or 37° C
Pathlength
1 cm
Mode
Kinetic
Reaction
Time
10 min.
Sample
Volume
100 µl
Reagent
volume
1 ml
Total
Volume
1.1 ml
Sample
to reagent ratio
1/10
5.2
INSTRUMENT
Any instrument capable of reading absorbance accurately with a sensitivity of 0.001 absorbance at 405 nm may be used. The band width should be 10 nm or less, stray light 0.5% or less, and the wavelength accuracy within 2 nm.
5.3
CALIBRATION
No routine reagent calibration is necessary as the acid phosphatase activity is calculated by use of the molar absorptivity of a-naphthol fast red TR complex which is taken as 12.9 at 405nm.
5.4 PROCEDURE FOR TOTAL
ACID PHOSPHATASE
5.4.1 Prepare the required volume of acid
phosphatase working reagent. (See
4.3 Reagent Preparation section.)
5.4.2 Add 1 ml of ACP
reagent.
5.4.3 Pipette 100 µl of serum to be assayed and incubate for 5 minutes at 30° C or 37° C.
5.4.4 Record the absorbance
(A1).
5.4.5
Record
the absorbance (A2) at the end of an additional 5 minutes.
5.5 CALCULATION AND RESULTS
FOR TOTAL ACID PHOSPHATASE
Note:
One unit of activity is defined as that amount of enzyme which will hydrolyze
one micromole of a-naphthyl phosphate per minute under conditions of the
assay
DA/5
min X assay volume X 1000
Total
ACP U/L = ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑--‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑-- = DA/5
min X 171
12.9 X sample volume X 5 X light path
DA/5
min = change in absorbance per 5 minutes at 405nm
assay
volume (ml) = 1.1 ml
sample
volume (ml) = 0.1 ml
12.9 = Millimolar absorptivity of a-naphthol-fast red TR complex at 405nm
light
path = length of light path (usually 1 cm)
1000
= Conversion of units per ml to units per liter
5
= Conversion of DA
per 5 min to DA
per 1 min
Example:
A1
= 0.159, A2 = 0.180
Total
ACP Activity (U/L) = (0.180 - 0.159) x 171 = 3.6 U/L
5.6 PROCEDURE FOR NON
PROSTATIC ACID PHOSPHATASE
5.6.1 Prepare the required volume of acid
phosphatase working reagent. (See
4.3 Reagent Preparation section.)
5.6.2 Add 1 ml of ACP reagent and 0.01 ml of
ACP tartrate reagent. Mix by
inversion.
5.6.3 Pipette 100 µl of serum to be assayed and
incubate for 5 minutes at 30° C or 37° C.
5.6.4 Record the absorbance
(A1).
5.6.5 Record the absorbance (A2) at the end of
an additional 5 minutes.
5.7 CALCULATION AND RESULTS
FOR PROSTATIC ACID PHOSPHATASE
Prostatic
ACP activity = Total ACP activity - Non Prostatic ACP
activity
Non
Prostatic ACP U/L =
DA/5
min X assay volume X 1000
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑------
= DA/5
min X 172
12.9
X sample volume X 5 X light path
DA/5
min = change in absorbance per 5 minutes at 405nm
assay
volume (ml) = 1.11 ml
sample
volume (ml) = 0.1 ml
12.9
= Millimolar absorptivity of a-naphthol-fast red TR complex at
405nm
light
path = length of light path (usually 1 cm)
1000
= Conversion of units per ml to units per liter
5
= Conversion of DA
per 5 min to DA
per 1 min
Example:
A1
= 0.151, A2 = 0.166
Non
Prostatic ACP Activity (U/L) = (0.166 - 0.151) x 172 = 2.6
U/L
Prostatic
ACP activity = 3.6 - 2.6 = 1.0 U/L
5.8 TEMPERATURE CONVERSION
FACTORS
To convert ACP activity from 30° C to 37° C, multiply the result by 1.33. To convert ACP activity from 37° C to 30° C, multiply the result by 0.76.
6.0 INTERPRETATION OF
RESULTS
6.1 EXPECTED VALUES
(10.4)
The range of expected values for adults (25-45 years)
is:
30° C
37°C
Total
ACP
2.2 ‑ 4.3 U/L
2.9 - 5.7 U/L
Prostatic
ACP
0.05 ‑ 0.9 U/L
0.07 - 1.2 U/L
Note:
ACP activity is age dependent.
These values are suggested guidelines. It is recommended that each laboratory establish the normal range for the area in which it is located.
6.2 MEDICAL ALERT VALUES
(10.11)
Each laboratory should establish low and high values beyond which the patient would require immediate attention by a physician. If a "medical alert value" is reached, always repeat the test to confirm the result and notify a physician if the result is confirmed.
6.3 LIMITATIONS OF
PROCEDURE
High levels of serum bilirubin reportedly inhibit acid phosphatase activity (10.9.) Oxalates and fluoride also inhibit the enzyme activity, while heparin and EDTA cause turbidity in the sample. Certain drugs and other substances are known to influence circulating levels of acid phosphatase (10.10.)
7.0 QUALITY
CONTROL
Standard practice for quality control should be applied to this system. Commercially available lyophilized controls can be used to monitor the daily acceptable variations. Normal and abnormal controls should be assayed at the beginning of each run of patient samples, whenever a new reagent or a different lot number is being used, and following any system maintenance.
A
satisfactory level of performance is achieved when the analyte values obtained
are within the "acceptable range" established by the
laboratory.
8.0 CALIBRATION
PROCEDURES
No routine reagent calibration is necessary as the acid phosphatase activity is calculated by use of the molar absorptivity of a-naphthol fast red TR complex which is taken as 12.9 at 405nm.
9.0 PERFORMANCE
CHARACTERISTICS
9.1
PRECISION
The
estimates of precision shown below were obtained from assays of human control
serum.
Within-Run
Samples
Mean (U/L)
SD (U/L)
CV (%)
15
2.86
± 0.17
5.9
15
18.4
± 0.29
1.6
Between-Run
Samples
Mean (U/L)
SD (U/L)
CV (%)
25
2.88
± 0.22
7.6
25
19.1
± 0.45
2.4
9.2
CORRELATION
A correlation study was done comparing this method and a similar acid phosphatase method. The samples range between 1.9 and 18.3 U/L.
Number
Regression Equation
Correlation
of Samples
y=Biotron, x=Comparative
Coefficient
28
y = 1.085 x - 0.422
0.995
9.3
LINEARITY
This procedure is linear through 40 U/L beyond which the specimen should be diluted 1 to 1 with isotonic saline. Reassay the specimen and multiply the results by 2.
9.4
SENSITIVITY
The
average sensitivity for this method is 0.006 DA/5
min per unit of concentration (U/L).
10.0
REFERENCES
10.1 Frankel S, Reitman S, Sonnenwirth
AC: Gradwohl's Clinical Laboratory Methods and Diagnosis, vol 1, 7th ed., CV
Mosby, St Louis (MO), 1970, p 115
10.2
10.3 Kind PRN, King EJ, J Clin Pathol
7:322, 1954.
10.4 Babson
10.5 Seligman AM, Chauncey HH, Nachlas
MM, Manheimer LH, Ravin HA, J Biol Chem 190:7, 1951.
10.6
10.7 Bais R, Edwards JB, Clin Chem
22:2025, 1976.
10.8 Cooper JDH, Turnell DC, Price CP,
Clin Chim Acta 126:297, 1982.
10.9 Shaw LM, Brummund W, Dorio RJ, Am J
Clin Pathol 68:57, 1977
10.10 Effects of Drugs on Clinical Laboratory Tests,
3rd ed. DS Young, Editor, AACC Press, Washington (DC),
1990
10.11 G.J. Kost, "Critical Limits for Urgent
Clinician Notification at U.S. Medical Centers"; JAMA, Feb. 2, 1990; Vol 263,
No.5, p.704
Rev 10/99