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MLAB 2401: Clinical MLAB 2401: Clinical ChemistryChemistryKeri Brophy-MartinezKeri Brophy-MartinezChapter 5: Assessment of Iron, Porphyrins and Others
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Specimen Requirements: Specimen Requirements: Iron StudiesIron Studies– Serum without anticoagulant– Plasma with heparin ( check
product insert)–Oxalate, citrate or EDTA binds Fe
ions, so they are unacceptable–Early morning sample preferred due
to diurnal variation– No hemolysis
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Iron Study/Profiles
% Fe Sa tura tionTo ta l Iron
T IBCx
100
3
Three Components◦ Total Iron ( serum )◦ TIBC◦ % Iron Saturation ( Fe Sat )
The Iron Saturation is a measurement of how “full” transferrin is
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Assessing Iron Levels and Assessing Iron Levels and FormsFormsDirectly measured
◦Iron◦Transferrin
Beta globulin formed in the liver Measured by the amount of iron it can
bind
◦Ferritin Best diagnostic test for IDA Acute phase reactant
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Assessing Iron Levels and Assessing Iron Levels and FormsForms• Indirect measure–TIBC (Total iron-binding capacity)• Measures the total amount of iron that
apotransferrin can bind• Can be expressed as a percentage(percent
saturation)• Ratio of serum iron to TIBC
• Increased–Late pregnancy– IDA–Following hemorrhage–Following destruction of liver cells
• Decreased–Decreased synthesis of transferrin– Increased loss of urine proteins
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Test Methodologies: IronTest Methodologies: Iron• Colorimetric Procedure–Separate Fe from transferrin with a
strong acid– Iron is reduced from ferrous(Fe3+) to
ferric(Fe2+) state–Addition of a chromogen creates a
colored compound–Measurement of colored product by
spectrophotometry
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Iron Reference RangesIron Reference Ranges
– Diurnal variation
– Men: 65-165 µg/dL
– Women: 45-160 µg/dL
–Decreased Levels• Decreased intake• Increased need• Increased loss
– Increased Levels• Increased
absorption• Hemolytic anemia• Lead poisoning• Pernicious anemia• Megaloblastic
anemia• Hepatitis
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Test Methodologies:TIBCTest Methodologies:TIBC
Pre-treatment and Colorimetric Method
1. Add Fe3+ to saturate binding sites on transferrin
2. MgCO3 is added to remove unbound Fe3+
3. Mixture is centrifuged and the supernatant tested using the serum iron methodology
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Reference RangesReference RangesTransferrin
◦200-360 mg/dLFerritin
◦Male: 20-250 ng/mL◦Female: 10-120 ng/mL
TIBC◦250-425 µg/dL
% saturation◦15-55
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Test Methodology: Test Methodology: HemoglobinHemoglobinElectrophoresis
◦Discussed in separate unit
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Test Methodology: Test Methodology: PorphyrinsPorphyrinsScreening tests
◦Urinary PBG◦Urinary ALA◦Urinayr porphyrins
Quantitative Assays◦URO◦PROTO◦COPRO
Serve to classify porphyrias
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Lab MethodsLab MethodsWatson-Schwartz for Urinary
PBG( porphobilinogen)◦Screen for acute intermittent
porphyria◦Specimen
Qualitative: fresh morning urine Quantitative: 24 hour collection
◦Reference Range <2 mg/daily
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Watson-SchwartzWatson-SchwartzPrinciple
◦ PBG + Ehrlich’s reagent results in a red-orange chromogen
◦ Interferences Urobilinogen indole
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Lab Methods: HgbA1cLab Methods: HgbA1cElectrophoresisEnzymatic AssaysHPLC
◦Goal is to separate hemoglobin forms within a column. Then, glycated versus total hemoglobin can be measured spectrophotometrically
◦Specimen requirements EDTA whole blood Can be non-fasting
Reference range◦4.0-6.0%
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Lab Methods: MyoglobinLab Methods: MyoglobinProcedures incorporate the
binding of specific antibodies to myoglobin with a resulting chemical or physical change that can be measured and correlated to myoglobin concentration
Specimen requirements◦Usually plasma ( check product
insert)
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Specimen Requirements: Specimen Requirements: LeadLeadWhole blood
◦Why? Circulating lead found in the RBC
◦Venous sample preferred but capillary sample can be used ( must confirm positive on capillary)
◦Royal blue top with EDTA anticoagulant
◦Lead-free containersUrine
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Lab Method: LeadLab Method: LeadTest methodologies
◦AAS◦Anodic stripping voltammetry
Reference Ranges in blood◦Children< 10 µg/dL