MLAB 2401: Clinical Chemistry Keri Brophy- M artinez

MLAB 2401: Clinical ChemistryKeri Brophy-Martinez Chapter 5: Assessment of Iron, Porphyrins and Others

Specimen Requirements: Iron 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

Three Components Total Iron ( serum ) TIBC % Iron Saturation ( Fe Sat ) The Iron Saturation is a measurement of how “full” transferrin is Iron Study/Profiles

Assessing Iron Levels and Forms • Directly 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 Forms • 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: 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 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:TIBC Pre-treatment and Colorimetric Method • Add Fe3+ to saturate binding sites on transferrin • MgCO3 is added to remove unbound Fe3+ • Mixture is centrifuged and the supernatant tested using the serum iron methodology

Reference Ranges • Transferrin • 200-360 mg/dL • Ferritin • Male: 20-250 ng/mL • Female: 10-120 ng/mL • TIBC • 250-425 µg/dL • % saturation • 15-55

Test Methodology: Hemoglobin • Electrophoresis • Discussed in separate unit

Test Methodology: Porphyrins • Screening tests • Urinary PBG • Urinary ALA • Urinayr porphyrins • Quantitative Assays • URO • PROTO • COPRO • Serve to classify porphyrias

Lab Methods • Watson-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-Schwartz • Principle • PBG + Ehrlich’s reagent results in a red-orange chromogen • Interferences • Urobilinogen • indole

Lab Methods: HgbA1c • Electrophoresis • Enzymatic Assays • HPLC • 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: Myoglobin • Procedures 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: Lead • Whole 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 containers • Urine

Lab Method: Lead • Test methodologies • AAS • Anodic stripping voltammetry • Reference Ranges in blood • Children< 10 µg/dL

MLAB 2401: Clinical Chemistry Keri Brophy- M artinez