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1-Pi deficiency in patients with lung diseases
1 Dept of Pneumology, University Hospital, Ruhrlandklinik and 2 Gemeinschaftspraxis Marx, Gelsenkirchen, Germany. 3 Biological Products, Bayer Corporation, Research Triangle Park, NC and 4 Heredilab Inc., and Dept of Internal Medicine, University of Utah, UT, USA
CORRESPONDENCE: M. Wencker, Schinkelstr. 44, 80805 München, Germany. Fax: 49 8932307789. E-mail: mwencker@aol.com
Keywords: 
1-Pi deficiency asthma, chronic obstructive pulmonary disease, gene frequency, screening
Received: October 27, 2001
Accepted March 11, 2002
The study was supported by a grant from Bayer Corp., Research Triangle Park, NC, USA. E.J. Campbell's work was supported by PHS grant HL 46440.
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Abstract |
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 TOP Abstract MethodsResultsDiscussionAcknowledgementsReferences |
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1-Pi deficiency. The aims of this study were to evaluate the accuracy of a new method for quantifying 1-Pi through phenotyping from dried blood spots (DBS) and to test the hypothesis that the screening of a population at risk increases the detection rate for severe 1-Pi deficiency.
The accuracy of phenotyping results from DBS was compared to conventional methods in a total of 555 individuals. In a prospective study 1,060 patients with chronic lung disease were screened for 1-Pi deficiency using DBS.
The validation of the phenotyping method from DBS showed an accuracy of 100%. Out of 1,060 tested patients, none had a severe PiZ deficiency and only 3 had PiSZ, whilst 36 (3.34%) individuals were identified as heterozygous for PiMS and 39 (3.68%) for PiMZ.
No patients with severe 1-Pi deficiency could be detected in this population and the frequency of PiMS or PiMZ detected was similar to that of the normal population. Thus, the screening of an unselected population of chronic obstructive pulmonary disease and asthma patients may not detect a large number of individuals with severe 1-Pi deficiency.
Patients with severe
1-Pi deficiency suffer from increased morbidity and mortality, which is mainly due to the development of chronic obstructive pulmonary disease (COPD) and pulmonary emphysema 1, 2. The major known risk factors are active and passive smoking. Patients who stop smoking have a better prognosis than those who do not 3. Thus, an early detection of 1-Pi deficiency would enable patients to make lifestyle changes to either prevent or postpone the development of functional impairment. However, in most European countries and the USA, <10% of the expected homozygous PiZ individuals are identified 4–6. Implementing effective screening efforts could increase the rate of detection and enable early preventive measures to be taken.
Several studies found between 1.9–17.8% of homozygous PiZ individuals in referral groups of patients with COPD 7–9 indicating that a targeted screening of patients with COPD could significantly increase the detection of homozygous PiZ individuals. On this basis, the World Health Organization (WHO) recommended that all patients with COPD and adults and adolescents with asthma should be screened once in their life for 1-Pi deficiency using a quantitative test 10.
The diagnosis of 1-Pi deficiency is based on the measurement of 1-Pi serum levels and isoelectric focusing (IEF). So far, large screening efforts for 1-Pi deficiency have been hampered by the need to draw intravenous blood and send it to the testing laboratory for both types of laboratory tests. Using dried blood spots (DBS) both for quantitative measurements of 1-Pi and for determination of the phenotype would obviate the need for refrigeration and simplify handling and shipping. However, the use of DBS in large-scale screening programmes has not been validated. Therefore, the aims of this study were: 1) to determine the accuracy of the new DBS test for 1-Pi concentrations and phenotyping and to assess the feasibility of using DBS in physicians' offices for screening purposes; 2) to evaluate the gene frequencies of deficiency alleles in a large population of patients with chronic lung disease; and 3) to determine whether applying the WHO recommendations for the screening of patients with COPD and asthma can increase the detection of patients with severe 1-Pi deficiency, an important prerequisite for large-scale screening of a target population.
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Methods |
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1-Pi deficiency using dried blood spots1-Pi deficiency, aliquots of 427 samples of ethylenediamine tetracetic acid (EDTA)-anticoagulated whole blood were allowed to dry onto filter paper. Both the EDTA-anticoagulated blood and the dried blood were then subjected to 1-Pi immunoassay as described below. Dried blood samples that yielded 1-Pi concentrations <20 µM and the respective plasma samples were further subjected to IEF.
Field test
For subsequent direct validation of the immunoassay and phenotyping procedures, individuals who attended a National Educational Conference of the Alpha-1 Association (Boston, MA, USA) (a population expected to include many individuals with 1-Pi deficiency) were offered an opportunity to simultaneously donate EDTA-anticoagulated whole blood obtained by phlebotomy and blood obtained by puncture of a distal finger pad for drying onto filter paper. A total of 128 subjects provided informed consent and participated in the study. The samples were analysed by immuno assay as described below. All DBS samples and the corresponding whole blood samples were phenotyped.
Testing for 1-Pi deficiency in selected practitioners' offices in Germany
A total of 7 physicians' offices took part in the screening. Four were general practitioners and three were pulmonary physicians. One of them treated a single patient with 1-Pi deficiency. Between March and June of 1999, patients with either COPD (chronic bronchitis, chronic obstructive bronchitis and pulmonary emphysema), asthma, or bronchiectasis were offered a test for 1-Pi concentrations and phenotype. Written informed consent was obtained and blood samples were collected on filter paper for further processing. Attached to the filter paper was a questionnaire on which physicians ticked boxes relating to smoking history, clinical symptoms and pulmonary diagnosis.
A total of 1,156 samples were received for evaluation. In 89 cases poor spotting of the blood on the filter paper prevented detection of both 1-Pi concentrations and phenotyping. In seven cases, there was enough material for the determination 1-Pi concentrations only. Thus, 1,060 cases were available for further analysis.
Application of blood onto filter paper and subsequent elution
Whole blood, either obtained from a distal finger pad with a lancet, a hyperemic earlobe, or an EDTA-anticoagulated blood specimen obtained by phlebotomy, was spotted onto no. 903 paper (Schleicher & Scheull, Keene, NH, USA) to completely saturate circles of half an inch in diameter imprinted on the paper. The papers were then air-dried at room temperature and stored at 4°C in separate envelopes to avoid cross contamination. Samples were shipped in batches once a week via overnight courier to the laboratory. For subsequent analysis, 1/8-inch diameter aliquots of paper containing dried blood were either punched from the filter paper by hand or were punched in duplicate directly into microtiter plates using a Delphia Plate Punch (Wallac Oy, Turku, Finland).
Immunoassay for 1-Pi
Completely automated immunoassays for 1-Pi in EDTA-anticoagulated blood were performed in multiwell plates, as described previously 11. For the immunoassay of blood dried onto filter paper, 1-Pi was eluted from the filter paper aliquots using a Tecan RSP 8051 ID robotic sample processor (Tecan US, Durham, NC, USA), using 0.01 M phosphate, 0.9 M NaCl, 0.05% Tween, 0.1 mg·mL–1 bovine serum albumin, 2.5% glycerol, pH 7.4 for 60 min, then diluted 1:17 into 96-well Fluoricon plates (IDEXX, Inc., Westbrook, ME, USA). The final multiwell plate included the following, each in duplicate: 1) a reference sample, 2) a normal control sample, 3) a sample from an individual known to have 1-Pi deficiency (PiZ), and 4) blank wells and four standards of known 1-antitrypsin concentration.
A completely automated particle-concentration immunoassay was then performed in a Screen Machine (IDEXX), essentially as described previously 11. Digital fluorescence data were transferred to microcomputers, which referenced the unknown samples to the standards and generated reports of the results.
Isoelectric focusing
For determination of 1-Pi phenotypes, samples of EDTA-anticoagulated whole blood were subjected to IEF in polyacrylamide gels (LKB Multiphor II and LKB Macrodrive 5 Constant Power Supply, Amersham Pharmacia Biotech, Piscataway, NJ, USA), essentially as previously described 12.
The 1-Pi phenotypes of samples of dried blood were determined by adding dithiothreitol to the eluted blood to a final concentration of 0.5 M, then subjecting the sample to IEF. Following IEF, proteins were passively transferred to nitrocellulose paper and incubated with rabbit 1-Pi, then with goat anti-human immunoglobulin (Ig)G (Sigma-Aldrich, St Louis, MO, USA). The 1-Pi was then visualised by staining with nitro blue tetrazolium 13.
Statistical methods
Data are presented as mean±sd. To compare differences between groups, a one way analysis of variance (ANOVA) was used. The Friedman test was used for variables that were not normally distributed. A Mann-Whitney test was performed for the comparison of two unrelated samples. In the analysis of the influence of different phenotypes on the 1-Pi concentration, the nonparametric Kruskal-Wallis H test was used. To assess the relationship of two categorical variables, the Chi-squared test was used. The Pearsons coefficient was calculated to analyse the linear correlation of two variables.
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Results |
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. The agreement between immunoassay results obtained from these two samples was excellent. Clinically significant 1-Pi deficiency is characterised by an 1-Pi serum concentration of <11 µM. All samples having 1-Pi concentrations <11 µM with the plasma immunoassay also had 1-Pi concentrations <11 µM with the immunoassay from the corresponding dried blood sample.
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1-Pi concentrations <20 µmol were phenotyped, all 29 individuals with levels <11 µM, but no others, had the deficient phenotype PiZ. Thus, the samples dried onto filter paper yielded no false-positives or false-negatives for 1-Pi deficiency, and all phenotypes determined from dried blood were identical to those determined from liquid blood.
Field study
Results of 1-Pi immunoassays of the 128 simultaneously drawn paired liquid and dried blood samples obtained at the National Educational Conference of the 1 National Alpha-1 Association are shown in figure 1.
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1-Pi concentrations was 0.951 (p<0.0001). All of the DBS samples with 1-Pi levels <11 µM, but no others, had 1-Pi concentrations <11 µM by standard immunoassay of liquid venous blood (table 1
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Individuals with an 1-Pi <11 µM, but no others, were found to have phenotype PiZ. In total there were 52 M, 7 MS, 36 MZ, 2 SZ and 26 Z individuals. Four subjects had the combination of an M allele and suspected rare allele on both phenotyping tests. One patient was on intravenous augmentation therapy with 1-Pi, and therefore no attempt was made to phenotype this individual. All phenotyping results on DBS were concordant with the corresponding venous blood samples.
Demographics of the screening population in Germany
A total of 1,060 individuals diagnosed with lung disease visiting a physician's office were screened for 1-Pi deficiency. Seventy-seven per cent of the patients (819 cases) were screened by pulmonary physicians; the remaining 241 were seen by general practitioners.
Four-hundred and ninety-eight (47%) were male and 548 (51.7%) were female. No information on sex was given in 14 individuals (1.2%). The mean age for the study group was 52.5±18.9 yrs (median 56.4 yrs, range 5–91 yrs). Eighty-seven individuals were <18 yrs and 217 were >70 yrs. Self-reported smoking history revealed 22.5% smokers, 33.0% ex-smokers, and 38.1% nonsmokers. Information was missing in the remaining 68 patients (6.4%). The mean age was higher in ex-smokers compared to nonsmokers and smokers (60.7±14.7 yrs versus 47.6 and 49.8 yrs, respectively, p<0.0001).
Dyspnoea on exertion was the most frequent complaint (69.3%), followed by cough (61.9%), phlegm (50%), asthma attacks (43.3%), and wheezing (42.1%). Only one-fifth of the patients presented with a single complaint, 22.4% had two symptoms that were listed on the questionnaire, 17.4% had three, 12.6% had four, and 27.1% had all five symptoms.
Asthma was the most frequent diagnosis made by the treating physicians in these patients and was present in 419 patients (table 2). In 89.9%, asthma was not accompanied by other diseases. As expected, asthma was the most frequent diagnosis in adolescents with a prevalence of 78% in the population tested. COPD was diagnosed in 390 patients, and in almost half of the cases this diagnosis was associated with other lung diseases. Pulmonary emphysema was found in 301 patients, and in 70.1%, other lung diseases were also present. Bronchiectasis was only diagnosed in 17 patients. In patients >70 yrs the prevalence of COPD with or without emphysema was 80%. In 158 patients, other diagnoses, mainly chronic bronchitis without obstructive lung disease, were present or information was missing.
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1-Pi concentrations of the 1,060 individuals tested are shown in table 3.
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1-Pi concentrations (p<0.0001). The highest were found in patients with PiM phenotype. They were significantly higher than for PiMS or PiMZ individuals (p<0.0001, both comparisons), and these again were significantly higher than for PiSZ (p<0.05, both comparisons). No homozygous PiZ and only three PiSZ individuals were found. The percentage of PiMZ for the three most commonly diagnosed lung diseases was 3.9% for COPD, 2.9% for emphysema and 2.9% for asthma, respectively. Patients with acute or chronic bronchitis as the only diagnosis were omitted from the analysis. The gene frequencies stratified for the diagnoses are shown in table 4.
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1-Pi deficiency S or Z for any given diagnosis. There was also no statistically significant difference between the gene frequencies for reported smoking status. |
Discussion |
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TOPAbstractMethodsResultsDiscussionAcknowledgementsReferences |
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1-Pi concentrations from DBS proved to be very reliable in comparison with conventional methods. In addition, phenotyping samples by a combination of IEF and immunoblotting from DBS showed an accuracy of 100%. In a prospective screening of patients with chronic lung disease, the gene frequency was similar to that found in normal volunteers 14–16. On the basis of this study the hypothesis that 2–3% of all patients with emphysema are homozygous for PiZ must be refuted in an unselected population seeking primary care for COPD and asthma.
DBS have been used in newborn screening to detect several diseases. They can be easily applied to a large number of individuals, no phlebotomy is required, and storage as well as transportation of samples does not require special containers or precautions. In the classic study by Laurell and Sveger 17, 108,000 Swedish infants were screened for 1-Pi deficiency using DBS. However, at the time they were still unable to quantify 1-Pi concentrations in dried blood and used the height of the transferrin peak as a relative reference. An enzyme-linked immunosorbent assay (ELISA) method similar to the one described in this study was first used in a screening of >39,000 newborns in Belgium 18 and proved to be a reliable method for quantification of 1-Pi. The 1-Pi protein and deoxyribonucleic acid collected by DBS is stable at room temperature over a period of 1 month 18. The method is simple and easily automated and therefore well suited for the screening of large numbers of samples. Recently, a Spanish group used eluates of DBS to quantify 1-Pi and determine the phenotypes. As in this study, the correlation of 1-Pi concentrations between DBS versus serum samples was excellent and a total concordance between the phenotyping results was observed 19.
To obtain results on the frequency of severe 1-Pi deficiency in a target population of patients with COPD or asthma seeking medical attention in physicians' offices, the current authors tested both for 1-Pi concentration and phenotype. This approach gave results for the rate of heterozygotes in the population and thus on the gene frequency of M, S and Z alleles. For the purpose of identifying individuals with severe deficiency only, a two-step approach would be more appropriate, in which it would be sufficient to phenotype individuals below a cut-off value of 20 µM 1-Pi in the serum only. In this case, a high percentage of heterozygous MZ and MS would not be detected. In this study, all samples were both quantitated and phenotyped. A cut-off of 20 µM would have identified all individuals with PiMO, S and SZ phenotypes. Of the carriers of one deficient allele, 11% of the PiMS and 56% of the PiMZ would have been identified. Thus a cut-off of 20 µM proved to be a safe method to identify all individuals with severe deficiency.
Several investigations in the 1970s reported a high prevalence of patients with severe 1-Pi deficiency in populations of patients with COPD and/or emphysema. In 1969, Lieberman and collegues 7, 20 investigated the serum trypsin inhibitory capacity (TIC) of 66 individuals admitted to a veterans hospital for pulmonary emphysema. On the basis of TIC alone, they identified 10.6% homozygotes and 15.2% heterozygotes; no phenotyping results were available. To study a possible selection bias by hospitals particularly interested in 1-Pi deficiency, investigators studied the phenotypes of 240 patients with COPD and/or emphysema in a referral population and compared them with those obtained by a hospital especially interested in 1-Pi deficiency 9. The percentages of both homozygous (2.5 versus 5.4% PiZ individuals) and heterozygous individuals (8.3 versus 13.8% PiMZ) in the specialised clinic were significantly higher, confirming a substantial selection bias induced by the specialisation in 1-Pi deficiency. Cox et al. 8 investigated 163 patients with COPD from the pulmonary service of a large urban hospital and found 4.9% PiZ patients in the whole group and as much as 17.8% PiZ in patients with pulmonary emphysema. In 1986, Lieberman et al. 7 found 1.9% homozygous PiZ individuals in a population of 965 patients with severe COPD. No details on the population tested were given. There is, however, evidence for a pronounced selection bias. Patients were severely impaired and were referred for bilateral carotid body surgery in a desperate attempt to alleviate dyspnoea.
Based on these reports, the WHO 10 stated that 2–3% of all emphysema patients were homozygous for PiZ and recommended screening for 1-Pi deficiency in patients with COPD and emphysema.
One of the aims of this study was to test the validity of the WHO recommendation for screening of populations at risk. The current authors' hypothesis was that by screening patients with lung disease, mainly COPD and emphysema, it would be possible to identify a large number of patients with severe 1-Pi deficiency. Using a more conservative estimate of 1–2% homozygous PiZ in the target population in this study would have led to the identification of 10–20 severely deficient individuals in 1,000 screened patients. The fact that no PiZ individuals in 1,060 screened patients were found in this study results in a frequency of homozygous PiZ of <0.1% as opposed to the 1–2% initially expected. Excluding patients with asthma from the analysis, there were 691 patients with COPD and/or pulmonary emphysema resulting in a frequency of homozygotes PiZ of <0.2%.
Even though there are obvious differences in patient selection between the historical studies cited above and the investigation presented here, the lack of severe 1-Pi deficient individuals in the population tested was unexpected. In contrast to the other studies, patients referred to a hospital where there is a particular interest in 1-Pi deficiency were not studied. Patients diagnosed as having COPD seeking primary care are likely to have less severe disease than patients hospitalised for COPD. In addition, the population of the previous studies was older and had a higher percentage of males. In this study lung function criteria were not used to define COPD. Even though most of the patients in this study had a lung-function test as part of their work-up, there may have been differences in the criteria for diagnosis of COPD. In addition, the use of TIC either alone or with acid starch electrophoresis might have led to an overestimation of the frequency of the Z allele. These factors point toward a significant selection bias in the historical studies investigating the percentage of PiZ individuals in a population of patients with emphysema. This study shows that these results cannot be extrapolated for a population of patients with COPD in a primary care setting and a large-scale screening of this patient group will not yield a 1–2% detection rate of homozygous PiZ. Targeted screenings following the recommendation of the WHO in its current form are not sufficient to increase the detection rate of 1-Pi deficiency. Additional selection criteria must be identified and evaluated for a high yield detection programme.
There has been considerable controversy regarding the increased risk of developing emphysema for patients with intermediate 1-Pi deficiency. Some studies have found an increased risk of development of COPD in individuals with PiMZ 8, 9, 21, while others have not 22, 23. In several population-based studies in Germany using IEF to determine the phenotypes, the percentage of PiMZ was 2.4–3.8%, which was not significantly different from the 3.7% found in this investigation 14–16. Although this implies that the frequency of heterozygous PiMZ is not increased in a population of patients with COPD, this study was not designed to test this hypothesis. A normal control group in the same geographical area being tested by the same laboratory methods would have been required to further investigate this question.
In conclusion, the dried blood spot method described in this paper is accurate and well suited for the screening of a large population. In contrast to previously published studies, an increased frequency of intermediate 1-Pi deficiency in patients with chronic obstructive pulmonary disease was not found. Since no individuals with severe 1-Pi deficiency were found, the percentage of patients with PiZ in an unselected population of patients with asthma or chronic obstructive pulmonary disease was <0.1%.
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Acknowledgements |
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References |
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1-antitrypsin deficiency. Acta Med Scand 1965;44:Suppl. 432, 1–85.
1-antitrypsin deficiency-related pulmonary emphysema with human 1-antitrypsin. Eur Respir J 1998;11:428–433.[Abstract]
1-Pi deficiency as a joint database of multiple national registries. Eur Respir J 1998;12:Suppl. 28, 382s–383s.
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1-antitrypsin deficiency. Am J Hum Genet 1975;27:213–217.[ISI][Medline]
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1-antitrypsin in dried blood spots from 39,289 newborns. Clin Chem 1991;37:821–825.This article has been cited by other articles:
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  G. Tirado-Conde, B. Lara, and M. Miravitlles Augmentation therapy for emphysema due to alpha-1-antitrypsin deficiency Therapeutic Advances in Respiratory Disease, February 1, 2008; 2(1): 13 - 21. [Abstract] [PDF]
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 M. Gorrini, I. Ferrarotti, A. Lupi, T. Bosoni, P. Mazzola, R. Scabini, I. Campo, M. Zorzetto, F. Novazi, and M. Luisetti Validation of a Rapid, Simple Method to Measure {alpha}1-Antitrypsin in Human Dried Blood Spots. Clin. Chem., May 1, 2006; 52(5): 899 - 901. [Full Text] [PDF]
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 C. de la Roza, F. Rodriguez-Frias, B. Lara, R. Vidal, R. Jardi, and M. Miravitlles Results of a case-detection programme for {alpha}1-antitrypsin deficiency in COPD patients Eur. Respir. J., October 1, 2005; 26(4): 616 - 622. [Abstract] [Full Text] [PDF]
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M. Dahl, C. P. Hersh, N. P. Ly, C. S. Berkey, E. K. Silverman, and B. G. Nordestgaard The protease inhibitor PI*S allele and COPD: a meta-analysis Eur. Respir. J., July 1, 2005; 26(1): 67 - 76. [Abstract] [Full Text] [PDF]
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 C P Hersh, M Dahl, N P Ly, C S Berkey, B G Nordestgaard, and E K Silverman Chronic obstructive pulmonary disease in {alpha}1-antitrypsin PI MZ heterozygotes: a meta-analysis Thorax, October 1, 2004; 59(10): 843 - 849. [Abstract] [Full Text] [PDF]
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: prolastin; 
: PiZ; •: PiMS; 
: PiM; 
: PiSZ; 
: PiMZ. DBS: dried blood spot.