Inorganic Phosphorus Reagent

Direct UV Method Without Reduction

PRODUCT SUMMARY SYMBOLS IN PRODUCT LABELLING
Stability : Until Expiry at 2-25°C Authorized Representative Temperature Limitation
Linear Range : 0.0 - 5.00 mmol/L (0 - 15.0 mg/dL) For in vitro diagnostic use Use by/Expiration Date
Specimen Type : Serum, plasma or urine Batch code/Lot number
Method : Endpoint CAUTION. CONSULT INSTRUCTIONS
Catalogue number FOR USE.
Reagent Preparation : Supplied ready to use.
Consult instructions for use Manufactured by

 Xi - Irritant

INTENDED USE specimens which have been refrigerated should be acidified and/or heated at 56°C for
This reagent is intended for the in vitro quantitative determination of Inorganic 15 minutes to redissolve any precipitate. (Acidified specimens are unsuitable for urate
Phosphorus in human serum, plasma or urine. or creatinine estimations). Dilute urine specimens 1 in 20 with distilled or deionized
water prior to analysis.
CLINICAL SIGNIFICANCE Storage: Serum or plasma samples are stable for 7 days at room temperature
More than 80% of the body’s phosphorus is present in bones as calcium (18-25°C) or 4°C and for 6 months frozen1. Urine samples when stored at 4°C are
phosphate. The remainder is found intracellularly as organic phosphates stable for 7 days. 6
such as phospholipids, nucleic acids and ATP or extracellularly as inorganic
phosphorus. ADDITIONAL EQUIPMENT REQUIRED BUT NOT PROVIDED
There is generally a reciprocal relationship between serum calcium and inorganic • If required, pipettes for accurately dispensing measured volumes.
phosphate levels. Increased levels of serum phosphorus are seen in renal • A clinical chemistry analyser capable of maintaining constant temperature (37°C)
disease, hypo-parathyroidism and excessive Vitamin D intake. Decreased and measuring absorbance at 340 nm.
levels are seen in rickets, osteomalacia (adult rickets), hyperparathyroidism • Analyser specific consumables, eg: sample cups.
and diabetic coma. 1 • Normal and abnormal assayed control material.
• Calibrator or a suitable aqueous Inorganic Phosphorus standard.
METHODOLOGY
Most methods for inorganic phosphate in serum involve the formation of ASSAY PROCEDURE
phosphomolybdate and subsequent reduction to molybdenum blue. Problems The following system parameters are recommended. Individual instrument applications
with variable color formation and reagent instability are common to these are available upon request from the Technical Support Group.
methods. 2,3
This procedure is based on the method of Daly and Ertingshausen4 as modified SYSTEM PARAMETERS
by Wang, et al. 5 The formation of the unreduced phosphomolybdate is measured Temperature 30°C / 37°C
at 340 nm and is directly proportional to the amount of inorganic phosphate Primary Wavelength 340 nm
present. Secondary Wavelength 380 nm
Assay Type Endpoint
REAGENT COMPOSITION Direction Increase
Active Ingredient Concentration Sample : Reagent Ratio 1 : 50
Ammonium molybdate 0.8 mmol/L eg: Sample Vol 3µL
Sulfuric Acid 430 mmol/L Reagent Vol 150µL
Sodium chloride 77 mmol/L Incubation Time 6 minutes
pH < 1.0 at 20°C. Reagent Blank Limits Low 0.0 AU
(340nm, 1cm lightpath) High 0.5 AU
WARNING: Do not ingest. If spilt, thoroughly wash affected areas with water. Linearity 0.0-5.00 mmol/L
Flush with plenty of water when disposing. For further information consult the (0-15.0 mg/dL)
Inorganic Phosphorus reagent Material Safety Data Sheet. Analytical Sensitivity 0.357 ΔA per mmol/L
R38 Irritating to skin. (340nm, 1cm lightpath) 0.115 ΔA per mg/dL
R41 Risk of serious damage to eyes.
S24/25 Avoid contact with skin and eyes. CALCULATIONS
Results are calculated, usually automatically by the instrument as follows:
REAGENT PREPARATION
Reagent is supplied ready to use. Absorbance of Unknown
Inorganic = ————————————— x Calibrator Value
Phosphorus Absorbance of Calibrator
STABILITY AND STORAGE
Prior to use:
When stored between 2-25°C and protected from light, the reagent is stable until Example:
the expiration date stated on the bottle and kit box label. Absorbance of calibrator = 0.496
Absorbance of unknown = 0.387
Once the reagent is opened: Value of calibrator = 1.39 mmol/L (4.31 mg/dL)
When stored capped between 2-25°C and protected from light, the reagent is
stable until expiry.
0.387
Inorganic Phosphorus = ———— x 1.39 = 1.08 mmol/L
Indications of Reagent Deterioration: 0.496
• Turbidity;
0.387
• Reagent Absorbance > 0.5 AU at 340 nm; and/or Inorganic Phosphorus = ———— x 4.31 = 3.36 mg/dL
• Failure to recover control values within the assigned range. 0.496

SPECIMEN COLLECTION AND HANDLING2 To convert the urinary phosphorus result from mmol/L to mmol/24 hours, the following
Serum: Use non-haemolysed serum. formula should be used:-
Plasma: Use heparin.
Urine: Accurate analysis of Urine phosphate can only beperformed if all Urine Phosphorus = Phosphorus Result x Dilution x Volume (L)
precipitated phosphate is dissolved prior to testing. Twenty Four hour urine (mmol/24 hours) (mmol/L) Factor
specimens should be acidified with 15 mL of concentrated HCl. Non acidified
 Example: PERFORMANCE DATA
Phosphorus = 1.55 mmol/L (4.81 mg/dL) The following data was obtained using the Inorganic Phosphorus reagent on a well
Dilution = 1 : 20 maintained automated clinical chemistry analyser. Users should establish product
Vol of Urine = 1.25 L (12.5 dL) performance on their specific analyser used.

Urine Phosphorus = 1.55 x 20 x 1.25 = 38.8 mmol/24 Hours IMPRECISION

Urine Phosphorus = 4.81 x 20 x 12.5 = 1203 mg/24 Hours Imprecision was evaluated over a period of 20 days using two levels of commercial
control and following the NCCLS EP5-T procedure. 10
NOTES
1. The sample and reagent volumes may be altered proportionally to LEVEL I LEVEL II
accommodate different spectrophotometer requirements. Number of data points 80 80
2. Specimens with inorganic phosphorus values greater than 5.00 mmol/L Mean (mmol/L / mg/dL) 0.70 / 2.17 1.04 / 3.22
(15.0 mg/dL) should be diluted and reassayed. Multiply the results by the Within Run: SD (mmol/L / mg/dL) 0.05 / 0.16 0.06 / 0.19
dilution factor. CV (%) 2.3 1.8
3. The color development is stable for at least 1 hour. Total: SD (mmol/L / mg/dL) 0.13 / 0.40 0.24 / 0.74
4. Unit conversion: mmol/L x 3.1 = mg/dL. CV (%) 6.2 7.5

CALIBRATION METHOD COMPARISON

Calibration is required. An aqueous standard or serum based calibrator, with Comparison studies were carried out using a similar commercially available Inorganic
an assigned value traceable to a primary standard (eg. NIST or IRMM) is Phosphorus reagent as a reference. Serum, plasma (Heparin) and urine samples
recommended. For calibration frequency on automated instruments, refer to were assayed in parallel and the results compared by least squares regression. The
the instrument manufacturer’s specifications. However, calibration stability is following statistics were obtained:
contingent upon optimum instrument performance and the use of reagents
which have been stored as recommended in the stability and storage section Serum/Plasma:
of this package insert. Recalibration is recommended at anytime if one of the Number of sample pairs 40
following events occurs:- Range of sample results 0.66 - 2.56 mmol/L
• The lot number of reagent changes. (2.05 - 7.95 mg/dL)
• Preventative maintenance is performed or a critical component is Mean of reference method results 1.23 mmol/L (3.8 mg/dL)
replaced. Mean of Inorganic Phosphorus results 1.19 mmol/L (3.7 mg/dL)
• Control values have shifted or are out of range and a new vial of control Slope 0.91
does not rectify the problem. Intercept 0.07 mmol/L (0.2 mg/dL)
Correlation coefficient 1.00
QUALITY CONTROL
To ensure adequate quality control, normal and abnormal controls should be Urine:
run as unknown samples:- Number of sample pairs 44
• At least once per day or as established by the laboratory. Range of sample results 2.9-62.6 mmol/L (9.0-194.0 mg/dL)
• When a new bottle of reagent is used. Slope 1.008
• After preventative maintenance is performed or a critical component is Intercept 0.32 mmol/L (1.0 mg/dL)
replaced. Correlation coefficient 0.9995
• With every calibration.
Control results falling outside the upper or lower limits of the established ranges LINEARITY
indicate the assay may be out of control. When run as recommended the assay is linear between 0 and 5.00 mmol/L
The following corrective actions are recommended in such situations:- (0 - 15.0 mg/dL).
• Repeat the same controls.
• If repeated control results are outside the limits, prepare fresh control serum ANALYTICAL SENSITIVITY
and repeat the test. When run as recommended the sensitivity of this assay is 0.357 ΔAbs per mmol/L or
• If results are still out of control, recalibrate with fresh calibrator, then repeat 0.115 ΔAbs per mg/dL (1 cm light path, 340nm).
the test.
• If results are still out of control, perform a calibration with fresh reagent, then REFERENCES
repeat the test. 1. Tietz N. W. (Ed), Textbook of Clinical Chemistry, W.B.
• If results are still out of control, contact Technical Services or the local Saunders Co. Philadelphia, 1986; p.1351-1354.
distributor. 2. Fiske C.H. and Subbarow U., J. Biol Chem, 66, 375 (1925).
3. Taussky H.H. and Schoor E.A.. J Bio Chem, 202, 675 (1953).
LIMITATIONS 4. Daly J.A. and Ertingshausen G., Clin Chem, 18, 263 (1972).
1. Studies to determine the levels of interference from haemoglobin, bilirubin 5. Wang J., Chen C.C., Osaki S., Clin Chem, 29 1255 (1983).
and lipaemia were carried out. The following results were obtained: 6. Shephard MD, Mezzachi RD. Clin Biochem Revs 1983; 4:61-7.
Haemoglobin: No interference from haemoglobin up to 160 mg/dL. 7. Young DS. Effects of Drugs on Clinical Laboratory Tests. Third Edition. 1990;
Bilirubin: No interference from bilirubin up to 340 µmol/L (20 mg/dL). 3:265-270.
Lipaemia: No interference from lipemia, measured as triglycerides, up to 8. Tietz Textbook of Clinical Chemistry and Molecular Diagnosis (4th Ed.) Burtis,
5.1 mmol/L (450 mg/dL). Ashwood & Bruns (Eds), Elsevier Saunders, 2005; 2290.
2. Young 7 has published a comprehensive list of drugs and substances which 9. Wachtel M et al, Creation and Verification of Reference Intervals. Laboratory
may interfere with this assay. Medicine 1995; 26:593-7.
10. National Committee for Clinical Laboratory Standards. User evaluation of
EXPECTED VALUES8 Precision Performance of Clinical Chemistry Devices. NCCLS; 1984, NCCLS
Serum: 0.8 - 1.5 mmol/L (2.5 - 4.5 mg/dL) Publication EP5-T.
24 hour Urine: 13 - 42 mmol/24 hr (0.4 - 1.3 g/24 hr)

The quoted values should serve as a guide only. It is recommended that each
laboratory verify this range or derive a reference interval for the population it
serves. 9

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