INORGANICPHOSPHORUS
INTENDEDUSE
METHOD
Onestepcolorimetricendpointmethodwithoutdeproteinization1,2
Vitroinorganicphosphorusreagentisintendedfortheinvitroquantitative
determination of inorganic phosphorus in human serum, plasma and
urineonbothautomatedandmanualsystems.
BACKGROUND
88%ofthephosphoruscontainedinthebodyislocalizedinboneintheformofcalciumphosphateastheapatiteCa2+(Ca3(PO))2 - 3.Theremainderisinvolvedinintermediary
carbohydrate metabolism and in physiologically important substances such as phospholipids, nucleic acids and ATP. Phosphorus occurs in blood in the form of inorganic
phosphateandinorganicallyboundphosphoricacid.Thesmallamountofextracellularorganicphosphorusisfoundalmostexclusivelyintheformofphospholipids.Theratioof
phosphate to calcium in the blood is approximately 6:10. An increase in the level of phosphorous causes a decrease in the calcium level. The mechanism is influenced by
interaction between parathormome and vitamin D. Hypoparathyroidism, vitamin D intoxication and renal failure with decreased glomerular phosphate filtration give rise to
hyperphosphatemia.Hypophosphatemiaoccursinrickets,hyperparathyroidismandFanconi’ssyndrome3.
ASSAYPRINCIPLE
The measurement of inorganic phosphorus in serum is usually accomplished by forming a phosphomolybdate complex and in turn reducing it to a molybdenum blue color
complex.Methodsdifferastothechoiceofreducingagents:stannouschloride4,phenylhydrazine5,aminonaphtholsulfonicacid6,ascorbicacid7,p-methylaminophenolsulfate8,Nphenyl-p-phenylenediamine9 and ferrous sulfate1. These methods suffered from color instability, deproteinization steps and complexity of performance8. Vitro reagent
eliminatedtheneedtoprepareaproteinfreefiltrate,acceleratedcolorproduction,stabilizedthecolorandsimplifiedtheprocedure.
1. Inorganicphosphorusreactswithammoniummolybdateinanacidicmediumtoformaphosphomolybdatecomplex.
2. Thiscomplexisreducedbyferrousammoniumsulfatetoproduceamolybdenumbluecomplex.
Acidicmedium
Inorganicphosphorus+ammoniummolybdate
Non-reducedphosphomolybdate
Non-reducedphosphomolybdate+Ferrousammoniumsulfate
Molybdenumbluecomplex
Theintensityofcolormeasuredphotometricallyat600-675nm,anditsintensityisdirectlyproportionaltoinorganicphosphorusconcentrationinthespecimen.
EXPECTEDVALUES
• Urinespecimensshouldbecollectedinacid-washedbottles.
• 24hoursspecimensshouldbecollectedincontainerscontaining5mlof
4.5–6.7mg/dl
• Inorganicphosphorusinacidifiedurinespecimensisstableifstoredat
Serum(fasting)10
Children(<2years)
6.0mol/lHCl
roomtemperature,refrigeratedorfrozen10.
(1.45-2.16mmol/l)
• Storedurinespecimenmustbemixedwellanddiluted1:10indistilled
4.5–5.5mg/dl
Children(2-12years)
waterprioranalysis.
(1.45–1.78mmol/l)
Adults(>12years)
Urine
(0.87–1.45mmol/l)
Onnonrestricteddiet
(12.9-42mmol/day)
Wavelength
Cuvette Temperature
Zeroadjustment
Specimen 0.4-1.3g/day
Each laboratory should investigate the transferability of the expected
valuestoitsownpatientpopulationandifnecessarydetermineitsown
referencerange.
REAGENTS
R1 Phosphorusstandard
R2 Sulfuricacid
Ferrousammoniumsulfate
Ferrousnitrate
Ammoniummolybdate
5.0 mg/dl
1.1
100
2.0
4.5
N
g/l
g/l
g/l
• ReagentPreparation&Stability
Allreagentsarestableuptotheexpirydategivenonlabelwhen
storedat2–8 0 C.
SPECIMEN
• Serum,plasma,andUrine.
• Theonlyacceptableanticoagulantisheparin.
SpecimenPreparation&Stability
• Serumorplasma
• Fresh serum collected in the fasting state is the preferred specimen
• Urine
MedicalDeviceSafetyServices
MDSSGmbH
Burckhardtstr.1
30163Hannover,Germany
Tel.:+4951162628630
Fax:+4951162628633
Blank
Standard
Specimen
1.0ml
R2
1.0ml
1.0ml
Standard
……
50 µl
……
Specimen
……
……
50 µl
User defined parameters for different auto analyzers are available
uponrequest.
CALCULATION
Calculatetheinorganicphosphorusconcentrationinserumbyusingthe
followingformulae:
SeruminorganicphosphorusConc.=
AbsorbanceofSpecimen
AbsorbanceofStandard
sinceseruminorganicphosphatelevelsareloweraftermeals.
600-675nm
1cmlightpath
20-250C
againstreagentblank
Serum,plasma,ordilutedurine
Mix, and Incubate for 15 minutes at room temperature Measure the
absorbanceofspecimen(Aspecimen)andstandard(Astandard)againstreagent
blank.
Thecolorisstablefor60minutes.
• AutomatedProcedure
XStandardvalue
• Unitconversion
mg/dlx0.323=mmol/l
• Use of anticoagulants other than heparin may interfere with the
formationofthephosphomolybdatecomplexandshouldnotbeused.
• Serum or plasma should be separated from blood cells as soon as
possible, to avoid the leakage of inorganic phosphorus and phosphate
estersintotheplasmamedia11.
• Inorganicphosphorusinserumisstable7daysrefrigeratedat40Cand
forthreeweekswhenfrozen9.
PROCEDURE
• ManualProcedure
2.5–5.0mg/dl
Calculate the inorganic phosphorus concentration in urine by using the
followingformulae:
UrineinorganicphosphorusConcentrationmg/24hrs=
AbsorbanceofSpecimen x5x10*xdxV
AbsorbanceofStandard
Where:
(5.0)inorganicphosphorusstandardconcentration.
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Article# DateofRevision
:133-EN
:10/2012
(10*)convertsmg/dltomg/l.
(d)dilutionfactor(10)
(V)the24hoursurinevaluesinliter.
QUALITYCONTROL
Itisrecommendedthatcontrols(normalandabnormal)beincludedin:
• Eachsetofassays,or
• Atleastonceashift,or
• Whenanewbottleofreagentisused,or
• Afterpreventivemaintenanceisperformedoraclinicalcomponent
isreplaced.
Commercially available control material with established inorganic
phosphorusvaluesmayberoutinelyusedforqualitycontrol.
Failuretoobtaintheproperrangeofvaluesintheassayofcontrolmaterial
mayindicate:
• Reagentdeterioration,
• Instrumentmalfunction,or
• Procedureerrors.
Thefollowingcorrectiveactionsarerecommendedinsuchsituations:
• Repeatthesamecontrols.
• If repeated control results are outside the limits, prepare fresh
controlserumandrepeatthetest.
• If results on fresh control material still remain outside the limits,
thenrepeatthetestwithfreshreagent.
• Ifresultsarestilloutofcontrol,contactVitroTechnicalServices.
INTERFERINGSUBSTANCES
•
•
•
•
•
•
•
MeanofInorganicPhosphorusresults3.7mg/dl
Slope0.91
Intercept0.2mg/dl
Correlationcoefficient1.00
Urine:
Numberofsamplepairs40
Rangeofsampleresults9.0-194.0mg/dl
Slope1.008
Intercept1.0mg/dl
Correlationcoefficient0.9995
Sensitivity
The sensitivity is defined as the change of analytical response per unit
changeinanalyteconcentrationatapathlengthof1cm.
Whenrunasrecommendedthesensitivityofthisassayis0.3mg/dl.
LINEARITY
Whenrunasrecommended,theassayislinearupto14mg/dl.
Specimenswithvaluesabove14mg/dlshouldbedilutedwith0.9%NaCl
solution or distilled water and reassayed. Multiply the result by the
dilutionfactor.
Anticoagulants:
The only acceptable anticoagulant is heparin. Complexing
anticoagulantsuchasEDTA,citrate,andoxalatemustbeavoided.
Bilirubin:
No significant interference from free or conjugated bilirubin up to a
level of 30 mg/dl. Interference may increase or decrease sample
results.
Haemolysis:
Avoid hemolyzed specimen since high amount of phosphate is
liberatedduringruptureoferythrocytes.
Lipemia:
Nosignificantinterference.
Drugs:
Young12 in 1990 has published a comprehensive list of drugs and
substances,whichmayinterferewiththisassay.
WARNING&PRECAUTION
BIBLIOGRAPHY
1. Taussky,H.etal.,(1953):J.Biol.Chem..202:675.
2. Goldenberg,H.,etal.,(1966):Clin.Chem.12;871.
3. Fraser et al., (1987): calcium and phosphate metabolism. In Tietz, N.W,
4.
5.
6.
7.
8.
9.
10.
11.
12.
Vitro phosphorous reagent is for in vitro diagnostic use only.
Normal precautions exercised in handling laboratory reagents
shouldbefollowed.
ed.Fundanintalofclinicalchemistry.3rded.Philadelphia:705-728.
Osmond,M.F.(1887):Bull.Soc.Chim.,Paris,47:745.
Taylor,AE.&Miller,CW(1914):J.Biol.Chem.,18:215.
Fiske,CH&Subbarow,YJ.(1925):Biol.Chem.66:375.
Lowry,OH.&Lopez,JA.(1946):J.Biol.Chem.162:421.
Power, MH. (1953): Standard methods of clinical chemistry, Academic
Press,NewYourk.
Dryer,RL.etal.(1957):J.BiolChem,225:177.
Tietz, N.W. ed, (1990): Clinical Guide to Laboratory Tests, 2nd
Philadelphia,Pa:WBSaunderscompany:444-447.
Weissman,N.&Pileggi,VJ(1974):inorganicions.In:HenryRJ,et.al.,eds
clinical chemistry principles and technics. 2nd ed. New York: Harper &
Row.720-728.
Young,DS(1990):EffectsofDrugsonClinicalLaboratoryTests.Third
Edition.1990:3:6-12.
Manufacturer
•
•
•
It is recommended that disposable PVC tubes be used for this
procedure.
Thereagentandspecimenvolumesmaybealteredproportionallyto
accommodatedifferentspectro-photometerrequirements.
Validresultsdependonanaccuratelycalibratedinstrument,timing,
andtemperaturecontrol
PERFORMANCECHARACTERISTICS
Imprecision
Reproducibility was determined using in an internal protocol. The
followingresultswereobtained.
WithinRun
BetweenDay
Control
LevelI
LevelII
LevelI
LevelII
Numberofsamples
40
40
40
40
Mean(mg/dl)
3.6
6.43
3.9
5.95
SD(mg/dl)
0.04
0.06
0.08
0.07
CV(%)
2.9
2.9
5.9
3.6
SYMBOLDECLARATION
Reagentisanacidandiscaustic.Avoidcontactwithskin.Flushwith
plentyofwaterifcontactoccurs.Don’tpipettebymouth.
REF
MethodComparison
Comparison studies were carried out using a similar commercially
available Inorganic Phosphorus reagent as a reference. Serum, plasma
(Heparin) and urine samples were assayed in parallel and the results
compared by least squares regression. The following statistics were
obtained:
ManufacturedinEgyptby:
TechnicalSupport:
+20226439699
VitroScient
www.vitroscient.com
[email protected]
Serum/Plasma:
Numberofsamplepairs40
©2012VitroScientCo
Rangeofsampleresults2.05-7.95mg/dl
Meanofreferencemethodresults3.8mg/dl
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Consultinstructionsforuse
Batchcode(Lot#)
Catalognumber
Temperaturelimitation
Invitrodiagnosticmedical
device
Useby
Caution.Consultinstructions
Keepawayfromlight
ORDERINGINFORMATION
SIZE
1341
2X50 ml
1342
2X100ml
orders:
+20226439698
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Article# DateofRevision
:133-EN
:10/2012