MLAB 2401: Clinical Chemistry
Keri 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
Iron Study/Profiles

Three Components
◦ Total Iron ( serum )
◦ TIBC
◦ % Iron Saturation ( Fe Sat )
 Total Iron 
% Fe Saturation  
 x 100
 TIBC 
 The Iron Saturation is a measurement of how “full”
transferrin is
3
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
– Decreased Levels
• Decreased intake
• Increased need
• Increased loss
– Increased Levels
–
Women: 45-160
µg/dL
•
•
•
•
•
•
Increased absorption
Hemolytic anemia
Lead poisoning
Pernicious anemia
Megaloblastic anemia
Hepatitis
Test 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 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