MLAB 2401: Clinical MLAB 2401: Clinical ChemistryChemistryKeri Brophy-MartinezKeri Brophy-MartinezChapter 5: Assessment of Iron, Porphyrins and Others

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

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

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

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

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

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

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

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

Test Methodology: Test Methodology: HemoglobinHemoglobinElectrophoresis

◦Discussed in separate unit

Test Methodology: Test Methodology: PorphyrinsPorphyrinsScreening tests

◦Urinary PBG◦Urinary ALA◦Urinayr porphyrins

Quantitative Assays◦URO◦PROTO◦COPRO

Serve to classify porphyrias

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

Watson-SchwartzWatson-SchwartzPrinciple

◦ PBG + Ehrlich’s reagent results in a red-orange chromogen

◦ Interferences Urobilinogen indole

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%

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)

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

Lab Method: LeadLab Method: LeadTest methodologies

◦AAS◦Anodic stripping voltammetry

Reference Ranges in blood◦Children< 10 µg/dL