PHOSPHORUS (FAST 340) PROCEDURE

Intended for the Quantitative Determination of Inorganic Phosphorus in Serum

SUMMARY AND EXPLANATION

Phosphorus plays an important role in amny physiological functions including intermediary matabolism, bone formation and acid-base balance (1). Increased serum phosphorus levels are seen with hypoparathyroidism, renal failure, hypervitaminosis and pyloric obstruction. Decreased serum phosphorus values result from hyperparathyroidism, myxedema, rickets and Fanconi Syndrone.Most methods for determining inorganic phosphorus employ the reaction of phosphate with molybdate and subsequent reduction of the phosphomolybdate complex to molybdenum blue (2). In 1972, Daly and Ertinghausen (3) described a procedure based on the measurement of the unreduced complex at 340 nm. Their method offered a more rapid analysis of serum phosphorus with improved reagent stability. The EAGLE PHOSPHORUS (FAST 340) PROCEDURE is based on this method.

PRINCIPLE

In an acid medium, molybdate reacts with inorganic phosphate to form phosphomolybdate. The absorbance at 340 nm is directly proportional to inorganic phosphorus concentration.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set  Cat. No. 01100 provides:

FAST PHOSPHORUS REAGENT - (Cat. No. 0111)

REACTIVE INGREDIENTS:

0.8 mM ammonium molybdate in an acid medium. Reaction accelerator added.

PRECAUTIONS:

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

STORAGE AND STABILITY:

Store at 15- 30ºC. Stable until expiration date.

DETERIORATION:

The reagent should be clear and colorless. A yellow-brown color would indicate deterioration, and the reagent should not be used. If the reagent blank has an absorbance greater than 0.300 at 340 nm, the reagent should not be used.

PHOSPHORUS CALIBRATOR - (Cat. No. 0112)

REACTIVE INGREDIENTS:

10 mg/dL Phosphorus as inorganic phosphate. Stabilizer added.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing.

STORAGE AND STABILITY:

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

DETERIORATION:

The calibrator should be clear and colorless. Turbidity would indicate deterioration, and the calibrator should not be used.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 340 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Fasting serum separated from the blood clot as soon as possible is recommended.

2. Plasma should not be used since anticoagulants may produce falsely low values.

3.      Hemolyzed samples may give falsely high values.

4. Jaundiced and lipemic samples require a serum blank.

SAMPLE STORAGE:

Phosphorus appears stable in serum for seven days at 2 - 8º C and for six months frozen.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Young et al, (4) have reviewed drug effects on serum inorganic phosphorus.

PROCEDURE

MATERIALS PROVIDED:

FAST 340 PHOSPHORUS REAGENT (Cat. No. 0111) and PHOSPHORUS CALIBRATOR (Cat. No. 0112).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.01 mL micropipettor

2. 1.00 mL pipettor

3. Test tubes, rack and timer

REACTION CONDITIONS:

Wavelength 340 nm

Reaction Type Endpoint

Incubation Time 1 minute

Incubation Temperature 15 - 30ºC

Sample Volume 0.01 mL

Reagent Volume 1.00 mL

Total Volume 1.01 mL

Low Normal 2.8 mg/dL

High Normal 5.0 mg/dL

Calibrator Value 10.0 mg/dL

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE: For instruments requiring a total volue of 1.0, increase PHOSPHORUS REAGENT volume to 2.0 mL and volume to 0.02 mL. Proceed as outlined.

1. Dispense 1.00 mL of FAST 340 PHOSPHORUS REAGENT into tubes labeled Reagent Blank, Control, Sample 1, etc.

2. Place 0.01 mL specimen into appropriate tube and mix well. Use D-H2O as specimen for Reagent Blank.

3. Permit the test sample to stand for 1 minute at room temperature.

4. Zero instrument at 340 nm using the Reagent Blank.

5. Read and record absorbances for Calibrator, Control and Unknowns.

NOTE: For a direct read-out instrument, set read-out at concentration of Standard and read the Unknown concentrations directly.

PROCEDURAL NOTE:

Linearity extends to 20 mg/dL. Specimens exceeding this value should be diluted with 0.9% sodium chloride solution and reassayed. Multiply test result by the dilution factor to obtain the final answer.

SERUM BLANK:

Jaundiced or lipemic samples requires a serum blank. Lypholized control sera are usually turbid after reconstitution and should also be blanked.

1. Dispense 1.00 mL of 0.9% sodium chloride solution into a tube.

2. Add 0.01 mL specimen. Mix well.

3. Zero instrument at 340 nm with 0.9% sodium chloride solution.

4. Read and record absorbance of the serum blank.

5. Subtract the serum blank absorbance from the sample absorbance obtained in STEP 5 of MANUAL PROCEDURE. Use this value in calculating the unknown concentration.

STABILITY OF THE FINAL REACTION PRODUCT:

The test samples should be read within 60 minutes.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0-20 mg/dL. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use PHOSPHORUS CALIBRATOR (Cat. No. 0112) which is provided in the reagent set for this purpose.

QUALITY CONTROL:

The realiability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for Unknowns. 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

The following equation is used to determine Unknown concentrations:

Unknown (mg/dL) =     

Unk. Abs.

------------- X Cal. Conc. (mg/dL)

Cal. Abs.

EXAMPLE:

A 10 mg/dL Calibrator had Abs. = 0.540 while the Unknown Abs. = 0.178. The unknown concentration of the Unknown is:

0.178 X 10 mg/dL

------------------------- = 3.3 mg/dL

0.540

LIMITATIONS

Detergents containing phosphate should not be used for cleaning glassware employed in this procedure.

EXPECTED VALUES (5)              

2.8 - 5.0 mg/dL

Phosphorus values vary greatly with the age of the patient. In infancy, normal values can be observed as great as 50% higher than in adults, gradually declining through childhood. After puberty, values in females tend to be approximately 10% higher than in males of the same age. It is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 20 mg/dL.

PRECISION:

Normal and abnormal control sera were assayed 20 times each for within run precision and for 10 working days to establish run to run precision.

WITHIN RUN:

MEAN / ST. DEV. / %CV

Normal 3.3 / 0.054 / 1.6

Abnormal 4.9 / 0.074 / 1.5

RUN TO RUN:

Normal 3.3 / 0.081 / 2.5

Abnormal 4.9 / 0.140 / 2.9

SPECIFICITY:

A comparison of this procedure with another widely used commercial method showed a 98% correlation.

SENSITIVITY:

This procedure has a sensitivity of 0.02 mg/dL per 0.001 absorbance unit.

REFERENCES

1. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd ed., W.B. Saunders Co., Philadelphia, 1976, p. 903.

2. Fiske, C.H., and Subbarow, Y., J. Biol. Chem., 66, 375 (1925).

3. Daly, J.A., and Ertingshausen, G., Clin. Chem., 18, 263 (1972).

4. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. Vol. 21, p.342D (1975).

5. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd ed., W.B. Saunders Co., Philadelphia, 1990, p. 1354.