How is the diagnosis of alpha-1 antitrypsin deficiency confirmed?

Diagnosis of Alpha-1-Antitrypsin Deficiency

The diagnosis of alpha-1-antitrypsin deficiency is confirmed by phenotyping using isoelectric focusing (IEF) of serum or plasma, which is the gold standard for identifying the specific AAT protein variant present. 1

Diagnostic Algorithm

The confirmation process follows a two-step approach:

Step 1: Quantitative Screening

• Measure serum AAT levels using nephelometry, radial immunodiffusion, or rocket immunoelectrophoresis 1
• Critical threshold: Levels <11 μmol/L (or <80 mg/dL by radial immunodiffusion, <50 mg/dL by nephelometry) indicate increased risk for emphysema 1
• Important caveat: AAT is an acute-phase reactant, so levels may be falsely elevated during inflammation, even in PI*ZZ patients 1
• A level ≥23 mmol/L (≥1.2 g/L) rules out severe deficiency in cases of moderate clinical suspicion, except for rare dysfunctional variants 2

Step 2: Qualitative Confirmation (The Gold Standard)

Phenotyping by isoelectric focusing (IEF) is the definitive confirmatory test 1:

• Separates AAT variants based on isoelectric point
• Requires skilled interpretation in reference laboratories
• Can be performed on serum, plasma, or dried blood spot samples (though dried spots should be confirmed with serum/plasma) 1
• Enhanced specificity when coupled with immunoblot or immobilized pH gradient IEF gel 1

Alternative/Supplementary Confirmatory Methods

Genotyping (DNA-based testing) 1, 2:

• Performed on genomic DNA from circulating mononuclear blood cells
• Detects known mutations (S and Z alleles) via allele-specific amplification
• Commercial kits available for whole blood or mouthwash samples
• Critical limitation: Will miss null alleles and rare variants
• DNA sequencing of SERPINA1 gene (exons 2-5) is proposed as the ultimate gold standard, particularly in high clinical suspicion cases 2

Key Clinical Pitfalls to Avoid

1. Never rely on AAT level alone for diagnosis 1:

   • Heterozygotes (PIMZ, PISZ) often have normal or borderline levels
   • Inflammatory conditions transiently increase levels even in severe deficiency
   • Commercial standards may overestimate concentration by 35-40% 1

2. Borderline levels (12-35 μmol/L or 90-140 mg/dL) require phenotyping 1:

   • May represent intermediate phenotypes (SZ, SS, MZ, MS)
   • Cannot be excluded based on quantitative testing alone

3. Liver biopsy findings are NOT diagnostic 1:

   • PAS-D positive inclusions >3 μm have only 94% specificity and 47% sensitivity for PI*Z allele
   • Can occur in other conditions (alcoholic hepatitis, viral hepatitis, hepatoma)
   • Phenotyping remains the gold standard even for liver disease diagnosis

When to Suspect and Test

The most recent Canadian guideline 2 and established ATS/ERS standards 1 recommend testing in:

• All COPD patients at diagnosis
• Adult-onset asthma with persistent airflow obstruction
• Unexplained bronchiectasis
• Early-onset emphysema (age ≤45 years)
• Emphysema with basilar predominance
• Unexplained liver disease (any age)
• Necrotizing panniculitis
• Family history of AATD, emphysema, or unexplained liver disease

Practical Testing Approach

For moderate clinical suspicion: Start with serum AAT level; if <23 mmol/L, proceed to phenotyping 2

For high clinical suspicion: Consider DNA sequencing as initial test, or collect both tubes (red cap for serum AAT, lavender cap for DNA) simultaneously 2

The diagnosis is definitively established when phenotyping identifies a deficient variant (most commonly PIZZ, but also PISZ, PI*SS, null variants, or rare variants like M malton or M duarte) in conjunction with low serum levels 1.