1. MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Chapter 5: Assessment of Iron, Porphyrins and Others

2. 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

3. Iron Study/Profiles 3 Three Components ◦ Total Iron ( serum ) ◦ TIBC ◦ % Iron Saturation ( Fe Sat )  The Iron Saturation is a measurement of how “full” transferrin is

4. 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

5. 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

6. Test Methodologies: Iron Colorimetric Procedure – Separate Fe from transferrin with a strong acid – Iron is reduced from ferrous(Fe 3+ ) to ferric(Fe 2+ ) state – Addition of a chromogen creates a colored compound – Measurement of colored product by spectrophotometry

7. 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

8. Test Methodologies:TIBC Pre-treatment and Colorimetric Method 1. Add Fe 3+ to saturate binding sites on transferrin 2. MgCO3 is added to remove unbound Fe 3+ 3. Mixture is centrifuged and the supernatant tested using the serum iron methodology

9. 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

10. Test Methodology: Hemoglobin Electrophoresis ◦ Discussed in separate unit

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

12. 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

13. Watson-Schwartz Principle ◦ PBG + Ehrlich’s reagent results in a red-orange chromogen ◦ Interferences  Urobilinogen  indole

14. 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%

15. 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)

16. 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

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