Estimation of iron profile

Estimation of iron profile

1. INSTITUTE OF PUBLIC HEALTH, DHAKA
Department of Laboratory Medicine
BSc in Health Technology (Laboratory)
Haematology
Estimation of Serum Iron (Fc)
By
Sk. MIZANUR RAHMAN
Assistant Bacteriologist,
PhD Candidate
MS in BGE; MS in Microbiology; MPH in Epidemiology

2. Objectives
To determine the normal level of serum iron.
To establish SOPs (Introduction, Procedure, QA, QC
etc.)
To correlate different reference value with interpretation
To determine the use of this test in diagnosis of anemia
(iron deficiency).

9. •Ironstudiesshouldbeperformedonfastingbloodsamplesbecause
recentingestionofironresultsinafalselyhighserumiron
concentrationand,therefore,transferrinsaturationlevel.
Special Notes
•The normalrangefortransferrinis 204 to 360 mg/dl. If you have a
higher amount, you may have iron-deficiency anemia. If you have a
lowerlevel, you may have another problem, such as liver disease and
hemolytic anemia.
•Transferrinmay also be measured using a value called total iron-
binding capacity (TIBC)
•Transferrinisablood-plasmaglycoprotein,whichplaysacentralrole
inironmetabolismandisresponsibleforferric-iondelivery.
•Itfunctionsasthemostcriticalferricpoolinthebody.
•Ittransportsironthroughthebloodtovarioustissuessuchastheliver,
spleenandbonemarrow.

10. Iron in the body
Iron is the metal component of
haemoglobin, myoglobin,
cytochromes and some proteins
of the electron transport chain.
The total iron of an adult male is
4-5g and of a female is 3-4g.

12. •Irondeficiencyisusuallydetectedonthebasis
oftheamountofironboundtotransferrininthe
plasma(serumiron)andthetotalamountofiron
thatcanbeboundtotheplasmatransferrinin
vitro
Normalvalues
Serumiron–80-180μg/dl
TIBC–250–370μg/dl
Serum Iron and Total Iron Binding Capacity

13. principle
Serumiron:TheirondissociatedfromitsFe-III-transferrin
complexbyadditionofacidicbuffercontaininghydroxylamine
whichreducestheFe(III)toFe(II).
Thenthechromogenicagent(PDTS)formahighlycolored
Fe(II)complexthatismeasuredspectrophotometricallyat
565nm.
UIBC:DeterminedbyaddingFe(II)toserumsothatitbinds
tounsaturatedironbindingsiteontransferrin.Theexcess
Fe(II)reactwithPDTStoformcolorcomplexwhichis
measuredspectrophotometricallyat565nm.Thedifference
betweentheamountofFe(II)addedandtheamountofFe(II)
measuredrepresenttheUIBC
TIBC:isdeterminedbyaddingserumirontoUIBCvalue.

14. principle

15. The serum iron gives a measure of the iron supply to the
tissues at the time of sampling.
Chromogenic Method
Principle
•WhenserumironistreatedwithMixedAcidReagent,the
proteinboundironisdetachedbytheactionofconc.HCl
&reducedtoferrousstatebythioglycolicacid&proteins
areprecipitatedbytheactionofTCA.
•Thisferrousironisthentreatedwithchromogensolution&
itformsacoloredcomplexwhichisthenmeasuredat562
nm.
Estimation of SerumIron

16. Estimation of SerumIron

17. Estimation of SerumIron

18. Estimation of SerumIron

19. 6. There should be no BT during last 20 days & no iron supplements taken
by the patient
Estimation of SerumIron

20. Method
Serum Iron
Blank Standard Test
Iron buffer
(pH 4.5)
2.5 ml 2.5 ml 2.5 ml
Iron
Standard
-------0.2 ml -------
Sample ----------------0.2 ml
Water 0.2 ml --------------
UIBC
Blank Standard Test
UIBC
buffer
2 ml 2 ml 2 ml
Iron
Standard
-------0.2 ml 0.2ml
Sample ----------------0.2 ml
Water 0.4 ml 0.2ml ------

21. Method

22. Calculations

23. Normal ranges
Serum iron (50 -160 μg/dl)
TIBC (250 -450 μg/dl)
Transferrin saturation (20 –55 %)

24. DefectinSerumiron
Serumironislowin-
irondeficiencyanaemiawhetherduetoinsufficientintake,
malabsorbtion,bloodlossorinabilitytoretrievestorageiron.
Serumironconcentrationishighwhen-
marrowcannotutilizeiron,hemolysis,increasedabsorptionor
defectsinstoragecapabilities.Highvaluesarealsofoundin
severehepatitisduetoreleasefromlivercells.
DefectinTotalironbindingcapacity(TIBC)
Increasein-
irondeficiencyanemia
Decreasein-
hemochromatosis,malignantorrheumaticfever.

25. Ferritinisfoundintracellularandinbloodstream.
Reliableparametertodeterminetheironstoresinthebody.
Itsdeterminationisanimportantparameterforthediagnosisandtherapy
controloftheirondeficiency.
Ferritinmeasurementisrecommendedforriskgrouplikeblooddonors,
pregnantwoman,hemodialysispatientandinfants.
Serumferritinreflectsbodyironstoresanditsmeasurementhasbeenwidely
usedasatestforirondeficiencyandironoverload
SERUMFERRITIN

26. Serum Ferritin can be measured by radio immunoassay or ELISA
( enzyme linked immunosorbent assay)
Most sensitive and specific test for diagnosis of iron deficiency
anaemia. Serum ferritin decreases even before the appearance of
anaemia
1 μg/L serum ferritin ≈10 mg storage iron
Serum ferritin < 12 μg/L is highly specific for diagnosis of iron
deficiency anaemia
SERUMFERRITIN

27. AntihumanFerritinAbareboundtomicro-wells.
FerritinpresentindilutedserumorpatientplasmabindtotherespectiveAb.
Washingofmicro-wellsremovesunspecificserumandplasmacomponents.
HRPconjugateantihumanferritinimmunologicallydetecttheboundpatient
ferritinformingaconjugate/ferritin/Abcomplex.
PRINCIPLE

28. Washingofthemicro-wellsremovesunboundconjugate.
Anenzymesubstrateinthepresenceofboundconjugatehydrolysestoformblue
color,theadditionofanacidstopsthereactionformingyellowendproduct.
Theintensityoftheyellowcolorismeasuredphotometricallyat450nm.The
amountofthecolorisdirectlyproportionaltotheamountofferritinpresentin
originalsample.
PRINCIPLE

29. MATERIALS

30. APPARATUS
1)Microplate
2)Multichanneldispenser
3)Laboratory timingdevice
4)Distilled or deionizedwater
5)Graduated cylinder 100 and 1000ml
6)Plastic container for the storage of washsolution.
7)Graphpaper.

31. 1)Prepare a sufficient no of micro plate wells to accommodate control and patient
samples.
2)Pipette 25 µl of calibrator, controls and patient samples in duplicate into the wells.
3)Add 100µl sample buffer to each wells.
4)Incubate for 30 min at 25⁰-28⁰ C.
5)Discard the content of micro-wells and wash 3 times with 300 µl of wash solution or
use automatic ELISA plate washer.
6)Dispense 100µl of enzyme conjugate into each well.
7)Incubate for 15 min at 25⁰-28⁰ C.
8)Discard the content of micro-well and wash
9)Dispense 100 µl of TMB substrate solution to each well
10)Incubate for 15 min at 25⁰-28⁰ C .
11)Add 100 µl stop solution to each well and incubate for 5 min at RT
12)Read the OD at 450 nm and calculate the result
PROCEDURE

34. Vitamin B12 and Serum Folate
TestIndications:
UsefulindetectingvitaminB12deficiencyanemia.Helpsdiagnosethe
causeofanemia,especiallywhentheRBC’saredescribedasmacrocyticin
non-neonates.HelpsdiagnosethecauseofdementiaorotherCNS
symptoms.

35. Notes
•VitaminB12andserumfolatearenolongerperformedonelderlypatientsin
theacutesettingunlessthereisaspecificindicatione.g.macrocytosis+/-
anaemia,pancytopenia,orbloodfilmreportcomment.
•Thistestwillnotberepeatedifithasbeencarriedoutwithinthelast3months.
•Thistestcanbeaddedontoapatientrequestifthelaboratoryhasan
appropriatesamplethatislessthan3daysold.
•Grosslyhaemolysed,lipaemicorictericsamplesmaygiveerroneousresults
andwillnotbeprocessed
•PatientPreparation:ForFolate:Patientsshouldbefasting(8hours
recommended)andshouldnothaverecentlyreceivedmethotrexateorother
folicacidantagonist.
Vitamin B12 and Serum Folate

36. TheVitaminB12assayprincipleisfortheRocheModularPchemistryanalyzer.
•Thetestisbasedontheprincipleofβ-complementationoftheenzymeβ-galactosidaseand
thecompetitionbetweenanenzymedonor-vitaminB12conjugate,ananti-vitaminB12
protein(IntrinsicFactor)andtheVitaminB12contentofaserumsample.
•SampleswithhigherVitaminB12concentrationsproducehigherβ-galactosidaseactivities
andviceversa.
•Anitro-phenyl-β-galactosidederivativeisusedastheenzymesubstrate.Thereaction's
producthasmaximumabsorbanceat415nm.
•TheVitaminB12concentrationofasampleisproportionaltothemeasuredβ-galactosidase
activity.
Vitamin B12-Assay Principle

37. Serum Folate
•Serumfolatefallsrapidlywithaninadequatedietdespitesufficient
bodystores.ItishowevermorereliablyanalysedthanRedCellfolate
andisthereforeusedtoscreenforfolatedeficiency.
•Thecommoncausesofdeficiencyareinadequatediet,malabsorption,
anti-folatedrugs(especiallyanticonvulsants)andoccasionallyin
associationwithanydiseasethatcausesincreasecellturnover,e.g.
exfoliation.
Result Interpretation

38. VitaminB12
Interpretationisdifficult.Thefollowingobservationsareoffered.
•Inclearcutdeficiency,levelsofB12arenearlyalways<150pg/mlandusually
<100pg/ml.
•Perniciousanaemiaisthecauseofthemajorityofseveredeficienciesinadults.
Around50%ofpatientswithperniciousanaemiahaveintrinsicfactorantibodies
whilethisantibodyisrarelyseeninnormalcontrols.Parietalcellantibodiesare
presentin90%ofpatientsbutarealsoseenin1-2%ofnormalcontrols.
•PatientswithB12intheborderlinerange(150-180pg/ml)mayhaveeitherearly
B12deficiencyorbehealthy"lownormal".
•Neurologicaldiseaseorglossitismayoccurwithoutanaemiaormacrocytosisand
maybeirreversible.
•Interpretationofearlymegaloblasticchangeinthemarrowisdifficultandcannot
bereliablydistinguishedfrommyelodysplasia.
Result Interpretation

39. VitaminB12:
PatientstakingvitaminB12supplementationmayhavemisleadingresults.Manyother
conditionsareknowntocauseanincreaseordecreaseintheserumvitaminB12
concentrationincluding:
Increases:IngestionofvitaminC,ingestionofestrogens,ingestionofvitaminA,
hepatocellularinjury,myeloproliferativedisorder,uremia.
Decreases:Pregnancy,aspirin,anticonvulsants,colchicine,ethanolingestion,
contraceptivehormones,smoking,hemodialysis,multiplemyeloma.Theevaluationof
macrocyticanemiarequiresmeasurementofbothvitaminB12andfolatelevels;
ideallytheyshouldbemeasuredsimultaneously.
Limitations

40. Folate:
Patientswithcombineddeficiencyoffolateandironmaynotdemonstratethe
erythrocytemacrocytosisotherwisetypicaloffolatedeficiencyanemia.Inthese
patients,however,theredcelldistributionwidth(RDW)willtypicallybeelevated.A
non-fastingspecimenresultsinfalselyelevatedresults.
Limitations