August 2009 Clinical Laboratory News: α1-Antitrypsin Deficiency


August 2009: Volume 35, Number 8

a1-Antitrypsin Deficiency
Stepping Up Awareness and Disease Detection
By Joshua Bornhorst PhD, DABCC


First described in 1963, α1-antitrypsin (AAT) deficiency is a potentially life-threatening autosomal genetic disorder that can lead to early-onset emphysema and/or liver disease. Today, recent estimates of the prevalence of AAT deficiency in the U.S. are as high as 1 in 3,000 individuals. Despite being one of the most common potentially lethal genetic diseases among caucasian adults, AAT deficiency often remains unidentified, in part because related pulmonary symptoms often do not manifest themselves until midlife when significant functional degradation has already occurred.

Genetic defects associated with AAT deficiency result in decreased circulating serum levels of AAT, leading to low alveolar concentrations, where it normally would serve as protection against proteases. In the absence of sufficient circulating active protease inhibitor AAT elastin, an alveolar structural protein, is degraded at an increased rate by the endogenous protease neutrophil elastase, leading to progressive pulmonary damage. In addition, select AAT protein variants are improperly processed during synthesis, forming aggregates in hepatocytes that can result in significant liver dysfunction.

Although awareness of this genetic disorder is increasing, the vast majority of individuals at risk for developing AAT deficiency-related disorders remain undiagnosed. Progression of disease can be retarded if AAT deficiency is identified; therefore, awareness of testing methods and algorithms for detection are important. This article describes the current understanding of AAT deficiency as well as testing methods and proposed algorithms for disease detection.

Genetics and Pathophysiology

AAT is the most abundant circulating protease inhibitor. The gene for the protein, known as SERPINA1, resides on chromosome 14 and contains one untranslated exon followed by four translated exons, resulting in a 394-amino acid gene product. While the AAT serine protease inhibitor irreversibly inhibits a number of different proteases, AAT is the main inhibitor of the neutrophil-derived protease elastase. In the absence of sufficient AAT protease, unopposed neutrophil elastase activity degrades the lung protein elastin and progressively damages the lower respiratory tract.

Like the gene associated with cystic fibrosis, the AAT gene locus is extremely polymorphic. In addition to the native M allele, which exhibits full anti-proteolytic activity, researchers have identified more than 100 unique variant alleles of the SERPINA1 gene. Individuals who possess two deleterious deficiency alleles are at substantial risk for developing symptoms related to AAT deficiency, and researchers believe the threshold for the occurrence of progressive pulmonary damage is a circulating serum level of < 50 mg/dL (or 11 μmol/L). This is approximately half of the lower limit of the reference interval serum concentrations in healthy individuals of 100 mg/dL.

The majority of pulmonary AAT deficiency related-disorders are caused by the inheritance of the two most common deficiency variants known as S and Z. The protein phenotype represented by the inheritance of two Z alleles, denoted here as PiZZ, results in a severe reduction of circulating AAT protease inhibitor. Heterozygous individuals, such as PiSZ, and to a lesser degree PiSS phenotypes, are also at some risk of developing AAT deficiency-related pulmonary problems, especially in the presence of certain aggravating environmental factors.

Researchers have also discovered a number of other less common AAT deficiency alleles. Individuals who inherit a combination of one of these rare allele variants and one of the common deficiency alleles can also have dangerously reduced circulating AAT levels (Table 1). In general, researchers currently believe that the inheritance of a single deficiency allele results in extremely low or no increased risk for pulmonary disorders.

Table 1
Common Phenotypes in AAT Deficiency


Approximate Percent of Normal Serum (AAT)

General Risk for AAT-Deficiency Related Clinical Disorders



Normal phenotype



None established



Possible slight risk of lung and liver disease



Some risk of lung disease



Elevated risk of lung disease and possible slight risk of liver disease



Substantial risk of lung and liver disease

*Other potential deficiency phenotypic variants include F, P, I, T and null. The degree of potential elevated risk of lung and liver disease in PiMZ individuals and liver disease in PiSZ individuals has not been established conclusively.

Undiagnosed individuals suffering from severe AAT deficiency usually develop emphysema and other pulmonary symptoms in the fourth to fifth decade of life, although symptoms may be observed earlier in smokers. The symptoms of AAT deficiency are similar to asthma or chronic bronchitis and patients can present with coughing, wheezing, persistent fatigue, and phlegm production. Patients also experience varying degrees of airflow obstruction, as exhibited by progressive reduced forced expiratory volume per second (FEV1). Chest radiographs may show evidence of hyperinflation, and lung function tests may show increased lung volumes. As a result of these problems, individuals suffering from severe AAT deficiency have reduced life expect-ancies.

Some deficiency variants, such as the Z mutant, are associated with liver disease, especially in individuals with the PiZZ phenotype. This is the result of AAT protein aggregates accumulating in the hepatocytes. The Z mutation induces an AAT protein conformation that is prone to polymerization, preventing protein export from the hepatocytes and protein degradation. The protein accumulates in the rough endoplasmic reticulum, in turn producing inclusion bodies that can be observed by periodic acid-Schiff staining in hepatic tissue samples. These inclusion bodies can trigger juvenile hepatitis, cirrhosis, and hepatocellular carcinoma.

However, the presentation of liver disease in individuals with the Z allele is variable. While some elevation of neonatal liver enzyme markers appear to be relatively common in PiZZ individuals, only 10% of these patients will exhibit neonatal hepatitis, with approximately 2% of all PiZZ individuals progressing to fibrosis or cirrhosis in childhood. Of PiZZ adult males, approximately one-third will develop cirrhosis, with an accompanying increase in the risk of developing hepatocellular carcinoma.

The association of other phenotypes that include a single Z allele with liver disease is less well defined. For example, controversy exists concerning a possible association of the PiMZ and PiSZ phenotype with slightly increased risk of developing liver disease. However, the S deficiency variant and the vast majority of rare AAT deficiency variants are not associated with increased risk of liver disease.

A number of other disorders have been linked with AAT deficiency. The potential development of panniculitis in individuals with AAT deficiency is well established. Evidence also exists that c-ANCA-positive vasculitis can be associated with AAT deficiency. Interestingly, researchers have recently shown that individuals with a heterozygous AAT deficiency phenotype exhibited a two-fold higher incidence of lung cancer than a control population. Future investigations may well link additional disorders to homozygous or heterozygous AAT deficiency variant phenotypes.

Common, But Not Well Known

While AAT deficiency is a relatively common disorder, most individuals who are at risk for developing AAT remain unidentified. As mentioned earlier, the prevalence of severe AAT deficiency is believed to be as high as 1 in 3,000 individuals in the U.S., making it one of the most common severe genetic diseases. The prevalence of the Z allele is highest in northern and western European populations, while the S allele is found most often in the Iberian Peninsula and southern European populations. In the U.S., as many as 3% of individuals are heterozygous for the Z allele, and an estimated one in 11.3 individuals have a (Pi)MS, MZ, ZZ, SZ, or SS phenotype, although the frequency varies by racial population. In addition, a host of other deficiency variants that occur at lower frequencies also have been observed in different populations. These include the null variants that produce little to no circulating AAT.

Today, approximately 100,000 PiZZ individuals likely reside in the U.S., yet fewer than 10,000 individuals have been diagnosed with AAT deficiency. According to estimates in several other industrialized nations, < 5% of individuals have been identified.

Treatment Options

Early diagnosis of AAT deficiency can slow or even arrest the progression of associated pulmonary disorders. Typically, affected individuals must alter their lifestyle and exposure to environmental factors and/or receive supplementation of circulating AAT protease inhibitor, known as augmentation therapy. Smoking and other environmental factors, such as excessive dust and fumes, appear to significantly impact the rate at which pulmonary function declines.

In particular, smoking increases the relative pulmonary elastase burden and reduces existing AAT protease inhibition activity by chemical oxidation. Furthermore, the decline in FEV1 in smokers relative to nonsmokers is more rapid. In siblings, emphysema has been observed at a higher frequency in smokers versus non-smokers.

Although more data are needed, augmentation therapy appears to slow the progression of the AAT deficiency-related pulmonary disease and reduce patient mortality rates. The therapy consists of infusions with purified AAT from pooled human serum. The American Thoracic Society (ATS) and the European Respiratory Society (ERS), have endorsed selective use of augmentation therapy. The potential benefits of the administration of such therapy must be balanced against its cost, which can be $50,000 or more per year. Work continues on developing additional treatment options for AAT deficiency.

Available Testing Options

Labs can choose from a number of methods to measure AAT levels in individuals with suspected disease. As the methodologies available for evaluation of AAT continue to evolve, the effectiveness of these individual assays or combinations of assays should continue to be evaluated.

Fifty years ago, researchers observed that patients with emphysema exhibited extremely low α1-globulin protein fractions by serum protein electrophoresis (SPEP). Modern automated electrophoretic techniques can provide incidental identification of patients in the course of routine SPEP testing for which further investigation of potential AAT deficiency should be considered, but the method is generally not suitable for investigating or screening for suspected AAT deficiency cases.

Today labs can measure serum protein levels directly by one of several commercially available nephelometric or immunoturbidimetric immunoassays. Low AAT serum levels provide a strong indication of potential AAT deficiency. While the use of serum to screen for AAT deficiency is relatively simple and inexpensive, some potential drawbacks exist. For example, normal intra-individual variation may limit the utility of detecting AAT serum levels in AAT deficiency. Alpha 1-antitrypsin is also an acute phase reactant and may be temporarily elevated due to a number of factors or conditions that can mask detection of AAT deficiency. Extensive liver disease or malnutrition can depress production of AAT. Finally, some rare deficiency variants exhibit relatively normal total serum concentrations but have reduced protease inhibition activity and are not effectively detected by serum concentration determination.

ATS and ERS currently consider protein phenotyping by isoelectric focusing as the gold standard method for observing protein variants associated with AAT deficiency alleles and diagnosing the disease. In this method, the AAT phenotype variants are separated by their isoelectric point on a thin layer polyacrylamide gel. While this analysis can identify both a large number of common and rare protein phenotypes, the assay and the interpretation of the resulting complex patterns can be technically challenging.

Labs can simplify variant detection with a commercial kit for isoelectric focusing that uses AAT-specific anti-serum (Sebia, Evry France). Aside from technical challenges, the drawbacks of all types of phenotyping by gel isoelectric focusing include similar relative migrations of different AAT phenotype variants with different underlying genetic variation and the inability to detect heterozygous null variants

Genetic testing can also play an important role in identifying individuals at risk for AAT deficiency. The most common molecular assays use rapid polymerase chain reaction (PCR) technology and specific multiplexed hybridization probes to reliably detect S and Z variants from a DNA sample. However, genotyping for the most common deficiency-associated alleles can miss significant numbers of at-risk individuals with other alleles such as null variants. In fact, some researchers estimate that approximately 5% of samples from individuals in a clinical population submitted for AAT testing that exhibit two putative deficiency alleles include at least one “rare” AAT deficiency variant. AAT deficiency in these individuals can go undetected when PCR assays for common deficiency S and Z alleles are used.

Researchers have also adapted genetic testing protocols to work with dried blood spots, facilitating more extensive targeted or screening efforts. Gene sequencing protocols have been described for all expressed AAT exon sequences; however, use of these technologies currently is limited in clinical practice due to the high cost and extended turnaround time. Denaturing gradient gel electrophoresis is another potential method for detecting nucleotide polymorphisms in the AAT coding and splicing sequences, but it is mainly used in research settings.

Testing Algorithms and Recommendations

When used in isolation, many individual testing methodologies are not completely sensitive for identifying AAT-deficient individuals. As discussed above, determination of serum levels is subject to multiple confounding factors that may cause a deficiency to be missed. In addition, there are technical and financial challenges inhibiting widespread availability of some assays. Consequently, a number of different types of testing algorithms have been proposed.

While further work needs to be done to compare and evaluate various test methodologies, algorithms combining multiple methodologies may prove to be the most effective and attractive option. Furthermore, a recently described automated assay for AAT antitryptic protease inhibitory activity may ultimately lead to additional alternative protocols for AAT-deficiency evaluation.

Figure 1 shows a flow chart of a generalized combined algorithm incorporating simultaneous serum concentration and rapid genetic testing for the S and Z alleles. In some cases, these methods are followed by protein phenotyping and further genetic testing. Other proposed algorithms include protocols in which initial screening for low serum concentration is performed and additional testing by phenotyping or genotyping is considered if indicated by low serum AAT concentrations.

Figure 1
Proposed Integrative Diagnostic Algorithm for AAT Deficiency

In this algorithm, the lab evaluates serum AAT levels by immuno-turbidimetric or nephlometric techniques and detects the S and Z deficiency alleles using a rapid PCR genotyping assay. Heterozygous genotypes for either the S or Z alleles are denoted as S Het or Z Het. Protein phenotypes are determined by isoelectric focusing (IEF).

Adapted from Synder et al. and Bornhorst et al.

ATS and ERS have developed testing recommendations for AAT deficiency in general populations like those of Western Europe and the U.S., as well as some proposed criteria (Table 2). The efficacy of targeted testing in select populations, such as individuals presenting with chronic obstructive pulmonary disease (COPD), has been validated. Such studies indicate that as many as 2% to 3% of individuals presenting with COPD are PiZZ individuals.

Table 2
Indications for Targeted Testing for AAT Deficiency

  • Symptomatic adults with emphysema, COPD, or asthma with airflow obstruction that is incompletely irreversible with bronchodilators who live in a geographic region with a relatively high prevalence of AAT deficiency, such as the U.S. or Northern Europe.
  • Asymptomatic individuals with persistent obstructive pulmonary dysfunction on testing who have a history of identifiable risk factors, such as smoking or occupational exposure.
  • Individuals with unexplained liver disease, including neonates, children, and adults.
  • Adults with necrotizing panniculitis.
  • Siblings of an individual with AAT deficiency.

Based on recommendations by the American Thoracic Society/ European Respiratory Society, Standards for the Diagnosis and Management of Patients with AAT Deficiency. Adapted from Stoller et al.(2007).

Suggested Readings

  • Aboussouan LS, Stoller JK. Detection of alpha-1 antitrypsin deficiency: a review. Respir Med 2009;103:335–41.
  • American Thoracic Society/European Respiratory Society statement: standards for the diagnosis and management of individuals with alpha-1 antitrypsin deficiency. Am J Respir Crit Care Med 2003;168:818–900.
  • Bornhorst JA, Procter M, Meadows C, Ashwood ER, et al. Evaluation of an integrative diagnostic algorithm for the identification of people at risk for alpha1-antitrypsin deficiency. Am J Clin Pathol 2007;128:482–90.
  • De Serres FJ. Alpha 1-antitrypsin deficiency is not a rare disease but a disease that is rarely diagnosed. Environmental Health Perspective 2003;111:1851–4.
  • Fairbanks KD, Tavill AS. Liver disease in alpha 1-antitrypsin deficiency: a review. Am J Gastroenterol 2008;103:2136–41; quiz 42.
  • Ferrarotti I, Scabini R, Campo I, Ottaviani S, et al. Laboratory diagnosis of alpha1-antitrypsin deficiency. Transl Res 2007;150:267–74.
  • Stoller JK, Aboussouan LS. Alpha1-antitrypsin deficiency. Lancet 2005;365:2225 –36. 9.
  • Stoller JK, Fromer L, Brantly M, Stocks J, et al. Primary care diagnosis of alpha-1 antitrypsin deficiency: issues and opportunities. Cleveland Clin J Med 2007; 74:869–74.
  • Snyder MR, Katzmann JA, Butz ML, Yang P, et al. Diagnosis of {alpha}-1-antitrypsin deficiency: an algorithm of quantification, genotyping, and phenotyping. Clin Chem 2006;52:2236–42.
  • Sveger T, Thelin T. A future for neonatal alpha1-antitrypsin screening? Acta Paediatr 2000;89:259–61.

Joshua Bornhorst, PhD, DABCC, is the director of chemistry, immunology, point-of-care, and neonatal clinical laboratories and assistant professor in the Department of Pathology at the University of Arkansas for Medical Sciences in Little Rock.



Page Access: