International Journal of Chronic Obstructive Pulmonary Disease Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Trends in Diagnosis of Alpha-1 Antitrypsin Deficiency Between 2015 and 2019 in a Reference Laboratory This article was published in the following Dove Press journal: International Journal of Chronic Obstructive Pulmonary Disease Irene Belmonte 1 Background: Alpha-1 antitrypsin deficiency (AATD) remains largely underdiagnosed Alexa Nuñez 1,2 despite recommendations of healthcare institutions and programmes designed to increase Miriam Barrecheguren 1 awareness. The objective was to analyse the trends in AATD diagnosis during the last 5 years Cristina Esquinas 1 in a Spanish AATD reference laboratory. Methods: This was a retrospective revision of all alpha-1 antitrypsin (AAT) determinations Mònica Pons 3 4 undertaken in our laboratory from 2015 to 2019. We analysed the number of AAT determi­ Rosa M López-Martínez nations performed and described the characteristics of the individuals tested, as well as the Gerard Ruiz 4 medical specialties and the reasons for requesting AAT determination. Albert Blanco-Grau 4 Results: A total of 3507 determinations were performed, of which 5.5% corresponded to Roser Ferrer 4 children. A significant increase in the number of AAT determinations was observed from 349 Joan Genescà 3,5 in 2015 to 872 in 2019. Among the samples, 57.6% carried an intermediate AATD Marc Miravitlles 1,6 (50–119 mg/dL) and 2.4% severe deficiency (<50 mg/dL). The most frequent phenotype in Francisco Rodríguez- severe AATD individuals was PI*ZZ (78.5%), and aminotransferase levels were above Frías 4 normal in around 43% of children and 30% of adults. Respiratory specialists requested the 1 Pneumology Department, Hospital highest number of AAT determinations (31.5%) followed by digestive diseases and internal Universitari Vall d’Hebron, Vall d’Hebron medicine (27.5%) and primary care physicians (19.7%). The main reason for AAT determi­ Institut de Recerca (VHIR), Vall d’Hebron nation in severe AATD adults was chronic obstructive pulmonary disease (41.7%), but Barcelona Hospital Campus, Barcelona, Spain; 2Universitat Autònoma de reasons for requesting AAT determination were not reported in up to 41.7% of adults and Barcelona, Bellaterra (Cerdanyola del 58.3% of children. Vallès). Barcelona, Spain; 3Liver Unit, Conclusion: There is an increase in the frequency of AATD testing despite the rate of AAT Department of Internal Medicine, Hospital Universitari Vall d’Hebron; Vall determination remaining low. Awareness about AAT is probably increasing, but the reason d’Hebron Institut de Recerca (VHIR), Vall for testing is not always clear. d’Hebron Barcelona Hospital Campus, Keywords: alpha-1 antitrypsin deficiency, diagnosis, screening, lung disease Barcelona, Spain; 4Department of Clinical Biochemistry, Hospital Universitari Vall d’Hebron, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; 5 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas Introduction y Digestivas, Madrid, Spain; 6CIBER de Alpha-1 antitrypsin deficiency (AATD) is an autosomal codominant hereditary Enfermedades Respiratorias (CIBERES), Barcelona, Spain disorder characterised by low serum levels of alpha-1 antitrypsin (AAT). At a clinical level, AATD is not properly a disease, but rather a predisposition for the development of pulmonary emphysema in adults and liver disease, especially in Correspondence: Marc Miravitlles children.1 Pneumology Department, Hospital AAT is a serine protease inhibitor encoded by the SERPINA1 gene. Over 150 Universitari Vall d’Hebron, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron mutations have been described in SERPINA1. According to the isoelectric focusing Barcelona Hospital Campus, P. Vall (IEF) pattern, the most common non-disease-causing allele is designated PI*M, and d’Hebron 119-129, Barcelona 08035, Spain Email marcm@separ.es the two most frequently deficient alleles (>95% of patients with AATD) are PI*S submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 2421–2431 2421 DovePress © 2020 Belmonte et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/ http://doi.org/10.2147/COPD.S269641 terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Belmonte et al Dovepress and PI*Z.2 The PI*Z variant, as well as other rare deficient phenotyping and genotyping of AATD samples from variants such as PI*Siiyama and PI*Mmalton can form other hospitals in Catalonia. The data analysed include intracellular polymers in hepatocytes, the primary source a 5-year period from January 2015 to December 2019. of AAT synthesis, leading to reduced levels of circulating The main objective of this study was to analyse the trends blood AAT.3,4 AAT levels in patients with the Pi*ZZ in AATD diagnosis during the last 5 years by measuring phenotype are approximately 15% that of normal values.1 the number of requests for AAT quantification in blood Laboratory testing for this deficiency comprises three samples received at the central core laboratory and their strategies: serum or plasma AAT quantification, AAT pro­ results in terms of blood concentrations as well as the tein phenotyping and genotyping.5 AATD is typically phenotype and/or genotype in samples demonstrating diagnosed by measurement of protein levels by nephelo­ AATD. A secondary objective was to explore the medical metry in routine blood analysis.5 If AAT quantification, specialities of the physicians sending samples and the corrected by inflammatory status measured by C-reactive reasons for AAT determinations. protein (CRP), is below the normal range, AATD should be confirmed by assessing the genotype, protein pheno­ Selection of Samples type, or a combination of both.5,6 All samples requesting AAT determination during the Guidelines from national and international societies, study period were included. In accordance to the results such as the World Health Organization,7 The Spanish of AAT quantification, samples were classified as follows Society of Pneumology and Thoracic Surgery8 and the based on the reference values of our laboratory: normal European Respiratory Society,1 recommend AAT testing, (AAT levels ≥120 mg/dL), intermediate deficiency (AAT at least once in a lifetime, for all patients with chronic levels between 119 mg/dL and 50 mg/dL) and severe obstructive pulmonary disease (COPD), emphysema, or deficiency (AAT levels < 50 mg/dL), as indicated by adults with asthma and irreversible airflow obstruction, Ferrarotti et al.13 All samples showing AAT deficiency and liver disease of unknown aetiology. were phenotyped by IEF using serum or plasma and the However, the number of patients diagnosed with Hydragel 18 A1AT Isofocusing kit (Sebia, Evry, France).14 AATD is much lower than expected according to epide­ Genotyping was performed in case of discrepancy between miologic studies9 and most patients are not correctly diag­ AAT levels and the phenotype observed. PI*S, PI*Z and nosed until late in the course of their pulmonary or liver PI*Mmalton alleles were genotyped by an allele-specific disease, after many visits to multiple physicians.10,11 genotyping assay using real-time polymerase chain reac­ Studies based on databases of medical records are tion and specific probes.15 Discordant cases were analysed increasingly used in clinical research, and since they repre­ by sequencing the entire encoding region of the sent real-life clinical practice, they may help to detect gaps SERPINA1 gene. Serum, plasma, whole blood or buccal or inadequacies in diagnosis and treatment.12 In this con­ swap samples were used indifferently for genotyping text, we performed the present study with the aim of methods.16 exploring the patterns of AATD diagnosis and their evolu­ Data about request origin and the reason for the request tion over the last 5 years. The results will help to design for AAT determination were collected, when available. programmes to increase awareness about the diagnosis of Since one of the main consequences of AATD is liver AATD. disease, we also tested some biochemical markers of liver damage available in routine blood analysis, such as alanine aminotransferase (ALT) and aspartate aminotrans­ Method ferase (AST) and calculated the Fibrosis-4 (FIB-4) score Study Design as (patient age x AST (IU/L))/(platelet count (x109/L) This was a retrospective review of all AATD determina­ x ALT−1/2 (IU/L)).17 The FIB-4 index can accurately dif­ tions carried out at the clinical laboratory of Vall d’Hebron ferentiate coinfected human immunodeficiency virus/hepa­ Barcelona Hospital Campus. This central core laboratory titis C virus patients with no or moderate hepatic fibrosis processes one of the largest number of samples of all (score < 1.45) from those with advanced fibrosis (score > laboratories in the Spanish public health system (around 3.25).18 A recent study showed that this index also has 6000 patients and 25,000 samples daily). Moreover, it is prognostic implications in patients with COPD.19 also a reference laboratory for AATD and performs However, the cutoff values for liver disease associated 2422 submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 DovePress Dovepress Belmonte et al with AATD remain undefined,20 and therefore, we used disease group, in addition to the clinical manifestations the score described above to classify our patients. described in adults, three AATD-associated complications As indications for AAT investigation may differ by which are clinically relevant in infants were added: chole­ age, the individuals studied were classified as children static hepatitis, poor feeding and poor growth. (<15 years of age) and adults (≥15 years old), according to the paediatric age in our centre, and were analysed Statistical Analysis separately. Descriptive analysis of all the samples collected during the study period was performed separately for children and Classification of Clinical Specialty of adults. Qualitative variables were described as absolute Requesting Physicians frequencies and/or the respective percentages. Mean and Clinical specialties requesting AAT determinations in standard deviation were calculated for quantitative vari­ adults were classified into 5 groups: 1) respiratory dis­ ables. Liver parameters, such as transaminases, and sam­ ease, 2) digestive disease and internal medicine, 3) pri­ ples with above normal levels were presented as multiples mary care, 4) external centres and 5) others. The of the upper limit of normal range (ULN) and percentages respiratory disease group included the departments of calculated based on the ranges established in our centre: pneumology, the respiratory system and cystic fibrosis. AST (10–35 U/L) and ALT (7–35 U/L) for adults and AST Digestive disease and internal medicine were included (15–60 U/L) and ALT (7–35 U/L) for children. FIB-4 has together because the Liver Unit is independent from the been validated in adults older than 18 years, and therefore, Digestive Diseases Department. This group also included it was calculated only in patients of this age group. Results specialists in the digestive system, gastroenterology, hepa­ are shown as percentages divided into three score ranges: tobiliary surgery and liver transplant units. The primary <1.45, 1.45–3.25 and >3.25. Comparisons of percentages care group included primary care centres of the Catalan among groups were performed by the Chi-squared test. Health Institute of the catchment area of our hospital. The A p value < 0.05 was considered significant. Statistical external centres group included other hospitals in analyses were performed with STATA version 14 Catalonia that sent samples to our laboratory because (StataCorp, College Station, TX, USA). AAT determination was not available. The remaining spe­ cialties were included in others. Results Regarding children, all the samples were classified Trends in AAT Determinations together in a group called paediatrics. A total of 3507 serum AAT determinations were per­ formed during the 5 years of the study, of which 192 Reason for Requesting AAT (5.5%) corresponded to children (Figure 1). Of these, Determination 2019 (57.6%) samples had intermediate AAT deficiency, The reasons for requesting AAT determination in adults with 1912 (54.5%) corresponding to adults and 107 (3.1%) were classified into 6 groups: 1) COPD, which included to children. The number of samples with severe deficiency COPD, emphysema or bronchitis; 2) bronchiectasis, con­ was 84 (2.4%), 72 (2.1%) of which were from adults and stituted by bronchiectasis and cystic fibrosis; 3) asthma; 4) 12 (0.3%) from children. liver disease, which included liver fibrosis, cirrhosis, hepa­ In general, the number of AAT determinations showed titis, liver failure, hepatocellular carcinoma (HCC), non- a gradual increase during the study period (Table 1 and alcoholic fatty liver disease, transaminase, bilirubin or Figure 2). As shown in Table 2, the clinical specialty with gamma-glutamyl transferase above reference values; 5) the highest increase was primary care, leading to a 5.4-fold others, when the requests were related to pathologies not increase regarding the number of AAT determinations associated with AATD; and 6) unknown. requested in 2015. Regarding children, the reasons for requesting AAT determination were divided into the same 6 groups, but Phenotype/Genotype of Samples with two slight differences: the COPD group was changed Of a total of 2103 deficient samples, 1978 (94%; 1899 to a group called bronchitis, which included bronchitis as intermediate and 79 severe deficiency) were phenotyped a reason of requesting AAT determination. In the liver and the remaining 125 (5.9%) could not be phenotyped/ submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 2423 DovePress Belmonte et al Dovepress Figure 1 Diagnostic algorithm followed in the study and number of samples obtained in each step. Abbreviations: AAT, alpha-1 antitrypsin; IEF, isoelectric focusing. genotyped due to insufficient or missing samples. Of the samples with severe deficiency (62, 78.5%), followed by 7 total of 1978 samples phenotyped, 254 (13%) were geno­ (8.9%) cases of the PI*SZ phenotype, 8 (10.1%) cases of typed, because the phenotyping results were not conclu­ rare or unknown mutations, 1 PI*SS and 1 PI*MM. The sive (Figure 1). PI*ZZ was the most frequent phenotype in severe deficiency sample (46.1 mg/dL) showing a PI*MM Table 1 Number of Alpha-1 Antitrypsin Determinations Performed, and Samples Detected with Deficiency by Year Period Total AAT Determinations Deficient Samples* Total Intermediate Deficiency Severe Deficiency Adults 2015 307 156 (50.8) 149 (48.5) 7 (2.3) 2016 520 306 (58.8) 298 (57.3) 8 (1.5) 2017 761 452 (59.4) 433 (56.9) 19 (2.5) 2018 888 546 (61.5) 530 (59.7) 16 (1.8) 2019 839 524 (62.5) 502 (59.8) 22 (2.6) Total adults 3315 1984 (59.8) 1912 (57.7) 72 (2.2) Children 2015 42 19 (45.2) 18 (42.9) 1 (2.4) 2016 44 26 (59.1) 25 (56.8) 1 (2.3) 2017 44 29 (65.9) 27 (61.4) 2 (4.5) 2018 29 20 (69) 18 (62.1) 2 (6.9) 2019 33 25 (75.8) 19 (57.6) 6 (18.2) Total children 192 119 (62) 107 (55.7) 12 (6.3) Total 3507 2103 (60) 2019 (57.6) 84 (2.4) Note: *Percentages (%) refer to the total AAT determinations performed each year. Abbreviation: AAT, alpha-1 antitrypsin. 2424 submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 DovePress Dovepress Belmonte et al Figure 2 Trends in alpha-1 antitrypsin determinations during the study period. phenotype was confirmed by sequencing of the four and the percentages for ALT were 48.6%and 34.6%, respec­ encoding exons of the SERPINA1 gene. This sample cor­ tively. However, the number of samples with increases >2 responded to a child with severe liver failure not related to ULN were only 4.5% for AST and 9.6% for ALT in adults AATD; and the PI*SS by phenotype (47.6 mg/dL), corre­ and 14.5% and 26.3% in children, respectively (Table 4). sponded to a sample from an external centre and could not The FIB-4 index was calculated in 2218 (66.9%) be genotyped due to insufficient sample. Regarding inter­ adults. Scores > 3.25 were observed in 3.5% of individuals mediate deficiency, 802 (42.2%) were PI*MM and 302 with normal AAT levels, 5.6% of those with intermediate (16%) PI*MZ. Interestingly, 68 (3.6%) had a rare or AATD (p=0.023 compared with normal AAT) and 9.3% of unknown genotype, and 10 (0.5%) were PI*ZZ. Out of individuals with severe AATD (p=0.031 compared with these 10 PI*ZZ, 9 were patients on augmentation therapy normal AAT) (Figure 3). Samples coming from requests of with AAT concentrations between 53 and 74 mg/dL; these specialists in digestive disease and internal medicine were samples were sent for evaluation of trough AAT serum those with a higher percentage of elevated aminotransfer­ levels. The remaining sample came from an external cen­ ase values and FIB-4 scores > 3.25 (Table 5). tre (77 mg/dL) and no more information could be obtained. The phenotypes of the adults and children with Clinical Specialties AATD are shown in Table 3. The highest number of requests was received from specia­ lists in respiratory diseases with 31.5% of total AAT Liver Parameters determinations, closely followed by specialists in digestive Serum aminotransferase quantification was requested in diseases and internal medicine with 27.5% and primary 2312 (69.7%) adults and 144 (75%) children. Up to 38.2% care physicians with 19.7% (Figure 4). Blood samples of children and 25.3% of adults had elevated AST levels, from paediatric patients had the highest diagnostic yield Table 2 Number of Alpha-1 Antitrypsin Determinations Performed by Each Clinical Specialty Every Year of the Study Period Clinical Specialty Total AAT Requested 2015 2016 2017 2018 2019 Respiratory disease 134 156 (1.2) 296 (2.2) 298 (2.2) 221 (1.6) Digestive disease and internal medicine 78 152 (1.9) 204 (2.6) 275 (3.5) 257 (3.3) Primary care 38 108 (2.8) 137 (3.6) 201 (5.3) 207 (5.4) External centres 45 80 (1.8) 79 (1.8) 63 (1.4) 111 (2.5) Paediatrics 42 44 (1) 44 (1) 29 (0.7) 33 (0.8) Others 12 24 (2) 45 (3.8) 51 (4.3) 43 (3.6) Note: Data are expressed as n (X-fold increase) regarding number of AAT determinations requested in 2015 by each clinical specialty. Abbreviation: AAT, alpha-1 antitrypsin. submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 2425 DovePress Belmonte et al Dovepress Table 3 Phenotypes Obtained in Adults and Children Classified into Intermediate and Severe Deficiency Phenotype Adults Children Intermediate Deficiency Severe Deficiency Intermediate Deficiency Severe Deficiency Common M/M 769 (42.8) – 33 (32.7) 1 M/S 527 (29.3) – 22 (21.8) – M/Z 282 (15.7) – 20 (19.8) – S/Z 72 (4) 7 14 (13.8) – S/S 77 (4.3) 1 5 – Z/Z 10 (0.6) 51 (76.1) – 11 (91.7) Rare M/Phe52dela 16 (0.9) – – – S/Phe52del 1 1 – – Z/Phe52del – 4 – – M/I 17 (0.9) – – – S/I 3 – – – M/PLowell 9 – 5 – S/PLowell 2 – – – Z/PLowell – 2 – – Z/F 1 M/Q0ourém 4 – – – M/Leu353Phe_fsX24b 1 M/Q0clayton 4 – 2 – M/Mvallhebron 1 – – – S/Ybarcelona 1 – – – Heterozygous u.m 1 1 – – Total 1798 67 101 12 a Notes: Data higher than 10 are expressed as n (%) regarding total phenotyped/genotyped samples of each deficiency group. Phe52del: Mmalton or Mpalermo variant (uncertain background); bLeu353Phe_fsX24: Q0mattawa or Q0ourém variant (uncertain background). Abbreviation: u.m, uncharacterized mutation. Table 4 Values of Serum Aminotransferases Expressed as Multiples of the Upper Limit of Normal and Classified into Normal AAT Levels, Intermediate or Severe Deficiency Adults Na ≤ 1.5 ULN > 1.5 ULN > 2 ULN > 3 ULN > 5 ULN AST ALT AST ALT AST ALT AST ALT AST ALT Normal AAT 818 765 733 53 85 27 52 15 28 6 12 Intermediate deficiency 1438 1300 1147 138 291 76 169 27 70 12 20 Severe deficiency 56 53 50 3 6 2 1 1 0 0 0 Children Normal AAT 53 35 25 18 28 16 22 11 18 5 11 Intermediate deficiency 79 71 59 8 20 3 12 3 6 2 2 Severe deficiency 12 10 8 2 4 2 4 2 2 2 1 Note: NaTotal of samples with aminotransferases in each deficiency group. Abbreviations: AAT, alpha-1 antitrypsin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal. for individuals with severe deficiency (6.3%), followed by requested by specialists in digestive diseases and internal samples referred by respiratory specialists, in which severe medicine. Table 6 shows the number of individuals char­ AAT was detected in 3.5% of individuals. Intermediate acterised by each clinical specialty and classified into AAT deficiency was found in 86% of AAT determinations normal levels, intermediate and severe deficiency. 2426 submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 DovePress Dovepress Belmonte et al and receives analytical requests corresponding to around 6000 patients daily. Although AAT testing is only recom­ mended once in a lifetime and the number of AAT determi­ nations increased along the study period, we consider that around 3500 requests in five years are a low number com­ pared to the number of samples processed in the lab and the clinical indications, which include highly prevalent diseases, such as COPD, adult asthma or liver disease. However, other large database studies have observed similar trends. Recently, Soriano et al21 analysed trends in AATD testing Figure 3 Percentage of adults with normal AAT levels, intermediate and severe and diagnosis in primary care between 1990 and 2014 in the deficiency divided into three score ranges of FIB-4 index: <1.45, 1.45–3.25 and >3.25. United Kingdom. They found the incidence of AATD diag­ Abbreviation: AAT, alpha-1 antitrypsin. nosis to have generally increased, but they also identified a low frequency of AATD testing, in which only 2.2% of Reasons for Requesting AAT patients diagnosed with COPD before the age of 60 years were tested. In our area, Barrecheguren et al22 identified an Determination increase in AATD testing in primary care over two periods In adults, documented lung disease (COPD, asthma and (2007–2008 and 2010–2011). However, the study showed bronchiectasis) was indicated as the reason for AAT determi­ that the number of AAT determinations performed was low, nation in 17.1% of samples, followed by liver (16.1%), and ranging from 4.33 per 10,000 inhabitants in 2007 to 6.85 per unknown with 41.7%. In contrast, in children, documented 10,000 in 2008, with no increase after 2008. Similar to our lung disease accounted for 7.9%, liver 27.1% and unknown results, the reasons for requesting AAT determination 58.3% (Figure 5). Samples from digestive and internal medi­ remained unclear in most cases, and only 13% of the adults cine had the highest frequency of unknown reasons (43%) and tested had AATD-associated respiratory diseases. samples from primary care the highest percentage of We observed a large increase in AAT determinations others (39%). during the study period with a total of 84 (2.4%) indivi­ duals being diagnosed with severe deficiency. Considering Discussion that the prevalence of severe AATD (specifically PI*ZZ) We report the trends in testing for AATD and the proportion among the general population in Spain is only 0.03%23 of different clinical specialties requesting AAT determina­ and among COPD patients in Europe it is 0.12%,24 tions during the last five years in a Spanish reference centre a detection rate of 2.4% for severe deficiency can be specialized in AATD diagnosis and treatment. The results considered a good diagnostic yield. Moreover, more than show that the number of AAT determinations performed in half (57.6%) of the individuals tested carried an intermedi­ our catchment area has significantly increased during the 5 ate deficiency, which is highly relevant for the individual years of the study. All specialties contributed to this increase, in order to prevent disease progression and to initiate but the largest increase was observed for samples from family screening for early detection of affected relatives. primary care. However, in most cases we could not identify Although AATD is one of the most common congenital the suspected diagnosis or the reason for requesting the test. disorders, it still remains significantly underdiagnosed.1,25 The Clinical Laboratory of the Vall d’Hebron Barcelona A number of reasons may explain this, including differ­ Hospital Campus is one of the largest laboratories in Spain ences in testing methods and the interpretation of results Table 5 Elevated Aminotransferases and FIB-4 Index Regarding the Clinical Specialties of Requesting Physicians Clinical Specialty AST >35 (U/L) ALT >35 (U/L) FIB-4 >3.25 Respiratory disease 95/790 (12%) 103/790 (13%) 18/770 (2.3%) Digestive disease and internal medicine 328/942 (34.8%) 442/942 (46.9%) 74/912 (8.1%) Primary care 124/438 (28.3%) 207/438 (47.3%) 6/402 (1.5%) Note: Data are expressed as number of samples with elevated liver parameters divided by total of samples for each clinical specialty. submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 2427 DovePress Belmonte et al Dovepress Figure 4 Percentage of alpha-1 antitrypsin determinations requested by different clinical specialties. and poor awareness of the disease. Studies from different During the study period, 1978 samples were phenotyped countries found low knowledge and unsatisfactory practice and/or genotyped. As expected, homozygous PI*ZZ made up related to AATD by physicians at all levels, from the largest number of individuals with severe deficiency, primary care to internal medicine specialists and followed by PI*SZ and heterozygous carrying Z/rare var­ pulmonologists.26,27 In a survey carried out in Spain and iants. In individuals with intermediate deficiency, the main Portugal,28 physicians reported referring patients to other phenotypes detected were PI*MM, PI*MS PI*MZ, and the specialists and the erroneous perception of the high cost of combination of a rare allele with the normal M variant was testing as the main reasons for not testing for AATD. the predominant rare phenotype observed. These results con­ However, the increasing requests for AATD diagnosis cur with the retrospective revision of 3511 AATD determina­ obtained in our study and others suggest that awareness tions performed in 2012, in which 1.6% of samples carried about AATD is probably increasing and barriers to AATD rare AAT alleles33 and among these variants, PI*I and testing are progressively being overcome. This might be PI*Mmalton represented 54% of cases. Interestingly, the due to more detection programs,29 the use of simpler number of rare variants detected increased in our study to testing approaches, such as buccal swaps or rapid point- 3.8% due to the higher number of genotyping tests per­ of-care testing,30 educational programmes provided by formed, but we observed exactly the same proportion of the reference centres and electronic alerts to encourage guide­ most frequent rare alleles PI*I and PI*Mmalton, which again line-based testing for AATD.31,32 accounted for 54.7% of all rare AATD alleles characterised. Table 6 Number of Alpha-1 Antitrypsin Determinations Requested by Each Clinical Specialty Divided into Normal AAT Levels, Intermediate and Severe Deficiency Clinical Speciality Total AAT Tests Requested Normal AAT Intermediate Severe Deficiency Deficiency Respiratory disease 1105 720 (65.2) 346 (31.3) 39 (3.5) Digestive disease and internal medicine 966 121 (12.5) 831 (86) 14 (1.4) Primary care 691 221 (32) 463 (67) 7 (1) External centres 378 195 (51.6) 174 (46) 9 (2.4) Paediatrics 192 73 (38) 107 (55.7) 12 (6.3) Others 175 74 (42.3) 98 (56) 3 (1.7) Note: Data are expressed as n (%) regarding total number of AAT determinations requested by each clinical specialty. Abbreviation: AAT, alpha-1 antitrypsin. 2428 submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 DovePress Dovepress Belmonte et al Figure 5 Percentage of reasons for requesting alpha-1 antitrypsin determination in (A) adults and (B) children. Serum aminotransferase levels were elevated in a large some heterozygotes that could show normal AAT serum number of samples; however, the number of samples with levels due to increased inflammation. clinically significant increases was below 10% in adults and around 25% in children, reflecting the different rea­ Conclusion sons for AAT determinations, with predominant liver dis­ Our study shows that the frequency of AATD testing is ease in children. The percentages of individuals with on the rise, despite the rate of AAT determinations in a FIB-4 index > 3.25 rose according to the severity of our large population (1.5 million inhabitants) remain­ AATD. These data suggest that FIB-4 might be sensitive to ing low. While the available data suggest that aware­ liver damage in AATD, but larger prospective studies are ness about AAT is probably increasing, physicians needed to confirm this.20 continue to face hurdles in the testing and diagnosis The Spanish Registry of Patients with Alpha-1 of patients. An increase in physician education about Antitrypsin Deficiency (REDAAT), mainly constituted AATD, regular reminders and the use of simpler testing by respiratory specialists, has recently established approaches would help to overcome the barriers to a diagnostic circuit with direct genotyping of buccal AAT testing. swaps.34,35 This circuit may have reduced the number of requests of blood analyses by respiratory specialists, which may account at least in part for the lack of Ethics Approval and Informed increase in the number of determinations during the Consent last year in our study. It is also of note that almost no The study was approved by the Ethics Committee of the samples were referred from family screening, because Vall d’Hebron Hospital (Barcelona, Spain). As this was most family screenings in our centre are conducted by a retrospective study and all samples were anonymized, direct genotyping from buccal swabs.34 informed consent was not considered necessary. Our study has some limitations: first, in many cases, we could not identify the reasons for requesting the determina­ Acknowledgment tion. Second, we have used a threshold of 50 mg/dL as Alexa Núñez is the recipient of a Rio Hortega contract in indicated by Ferrarotti et al13 and as used in previous reports the 2019 Strategic Action Health Call from the Instituto de by our group;5,22 however, this threshold is arbitrary, and Salud Carlos III for the years 2020–2022. Miriam other authors have suggested values around 57 mg/dL. Barrecheguren is the recipient of a Rio Hortega contract Third, we could not correct AAT serum levels by level of in the 2017 Strategic Action Health Call from the Instituto inflammation demonstrated by simultaneous quantification de Salud Carlos III for the years 2018–2019. Mònica Pons of CRP.36–39 With our diagnostic protocol, all PI*ZZ or null is the recipient of a Rio Hortega contract in the 2018 or rare alleles would be identified, irrespective of the simul­ Strategic Action Health Call from the Instituto de Salud taneous determination of CRP levels, but we might miss Carlos III for the years 2019–2020. submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 2429 DovePress Belmonte et al Dovepress Funding 10. Lara B, Blanco I, Martínez MT, et al. Spanish registry of patients with alpha-1 antitrypsin deficiency: database evaluation and popula­ This study was supported through funding from Grifols to tion analysis. Arch Bronconeumol. 2017;53:13–18. doi:10.1016/j. the Catalan Center for Research in Alpha-1 antitrypsin arbres.2016.05.003 11. Stoller JK. Detecting alpha-1 antitrypsin deficiency. Ann Am Thorac Soc. deficiency of the Vall d’Hebron Research Institute 2013;13(Suppl4):S317–S325. doi:10.1513/AnnalsATS.201506-349KV (VHIR) in the Vall d’Hebron Barcelona Hospital 12. Miravitlles M, Monteagudo M, Solntseva I, Alcázar B. Blood eosi­ Campus, Barcelona, Spain. nophil counts and their variability and risk of exacerbations in COPD: a population-based study. Arch Bronconeumol. 2020;12:pii: S0300–2896(19)30623–4. doi:10.1016/j.arbres.2019.12.015 Disclosure 13. Ferrarotti I, Thun GA, Zorzetto M, et al. Serum levels and genotype distribution of α1-antitrypsin in the general population. Thorax. Miriam Barrecheguren has received speaker fees from 2012;67:669–674. doi:10.1136/thoraxjnl-2011-201321 Grifols, Menarini, CSL Behring, GSK and consulting fees 14. Zerimech F, Hennache G, Bellon F, et al. Evaluation of a new Sebia from GSK, Novartis, Boehringer Ingelheim and Gebro isoelectrofocusing kit for alpha 1-antitrypsin phenotyping with the hydrasys system. Clin Chem Lab Med. 2008;46(2):260–263. Pharma, outside the submitted work. Cristina Esquinas has doi:10.1515/CCLM.2008.036 received speaker fees from CSL Behring. Marc Miravitlles 15. Belmonte I, Montoto L, Miravitlles M, et al. Rapid detection of mmaltonα1-antitrypsin deficiency allele by real-time PCR and melt­ has received speaker or consulting fees from AstraZeneca, ing curves in whole blood, serum and dried blood spot samples. Clin Bial, Boehringer Ingelheim, Chiesi, Cipla, CSL Behring, Chem Lab Med. 2016;54:241–248. doi:10.1515/cclm-2015-0297 Laboratorios Esteve, Gebro Pharma, Kamada, 16. Belmonte I, Barrecheguren M, Esquinas C, Rodriguez E, Miravitlles M, Rodriguez-Frias F. Genetic diagnosis of GlaxoSmithKline, Grifols, Menarini, Mereo Biopharma, alpha1-antitrypsin deficiency using DNA from buccal swab and Novartis, pH Pharma, Rovi, TEVA, Spin Therapeutics, serum samples. Clin Chem Lab Med. 2017;55(9):1276–1283. Verona Pharma and Zambon, personal fees from Sandoz doi:10.1515/cclm-2016-0842 17. Sterling RK, Lissen E, Clumeck N, et al. Development of a simple and Ferrer, and research grants from GlaxoSmithKline and non-invasive index to predict significant fibrosis patients with HIV/ Grifols, outside the submitted work. The aforementioned HCV co-infection. Hepatology. 2006;43:1317–1325. doi:10.1002/ hep.21178 authors report no other potential conflicts of interest for this 18. Vallet-Pichard A, Mallet V, Nalpas B, et al. FIB-4: an inexpensive and work. The remaining authors report no conflicts of interest. accurate marker of fibrosis in HCV infection. Comparison with liver biopsy and fibrotest. Hepatology. 2007;46(1):32–36. doi:10.1002/ hep.21669 References 19. Yong SH, Leem AY, Kim YS, et al. Hepatic fibrosis assessed using 1. Miravitlles M, Dirksen A, Ferrarotti I, et al. European respiratory fibrosis-4 index is predictive of all-cause mortality in patients with society statement: diagnosis and treatment of pulmonary disease in chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon alpha-1 antitrypsin deficiency. Eur Respir J. 2017;50:1700610. Dis. 2020;15:831–839. doi:10.2147/COPD.S242863 doi:10.1183/13993003.00610-2017 20. Strnad P, McElvaney NG, Lomas DA, Longo DL. Alpha1-antitrypsin 2. Greene CM, Marciniak SJ, Teckman J, et al. α1-antitrypsin deficiency. deficiency. N Engl J Med. 2020;382(15):1443–1455. doi:10.1056/ Nat Rev Dis Primers. 2016;16052. NEJMra1910234 3. Lomas DA, Finch JT, Seyama K, Nukiwa T, Carrell RW. Alpha 21. Soriano JB, Lucas SJ, Jones R, et al. Trends of testing for and 1-antitrypsin Siiyama (Ser53→Phe). Further evidence for intracellular diagnosis of α1-antitrypsin deficiency in the UK: more testing is loop-sheet polymerization. J Biol Chem. 1993;268:15333–15335. needed. Eur Respir J. 2018;52(1):1800360. doi:10.1183/ 4. Lomas DA, Elliott PR, Sidhar SK, et al. Alpha 1-antitrypsin mmalton 13993003.00360-2018 (Phe52-deleted) forms loop-sheet polymers in vivo. Evidence for the 22. Barrecheguren M, Monteagudo M, Simonet P, et al. Diagnosis of C sheet mechanism of polymerization. J Biol Chem. alpha-1 antitrypsin deficiency: a population-based study. Int J Chron 1995;270:16864–16870. doi:10.1074/jbc.270.28.16864 Obstruct Pulmon Dis. 2016;11:999–1004. 5. Miravitlles M, Herr C, Ferrarotti I, et al. Laboratory testing of indivi­ 23. Blanco I, de Serres FJ, Fernandez-Bustillo E, Lara B, Miravitlles M. duals with severe AAT deficiency in three European centres. Eur Estimated numbers and prevalence of PI*S and PI*Z alleles of Respir J. 2010;35:960–968. doi:10.1183/09031936.00069709 alpha1-antitrypsin deficiency in European countries. Eur Respir J. 6. McElvaney NG. Diagnosing α1-antitrypsin deficiency: how to 2006;27:77–84. doi:10.1183/09031936.06.00062305 improve the current algorithm. Eur Respir Rev. 2015;24:52–57. 24. Blanco I, Diego I, Bueno P, Pérez-Holanda S, Casas-Maldonado F, doi:10.1183/09059180.10010814 Miravitlles M. Prevalence of alpha-1 antitrypsin genotypes in patients 7. Barker A, Brantly M, Campbell E, Carrell R, Cox DW, Dirksen AE. with COPD in Europe: a systematic review. Eur Respir Rev. Alpha 1-antitrypsin deficiency: memorandum from a WHO meeting. 2020;29:200014. doi:10.1183/16000617.0014-2020 Bull World Health Organ. 1997;75(5):397–415. 25. Meira L, Boaventura R, Seixas S, Sucena M. Alpha-1 antitrypsin 8. Casas F, Blanco I, Martinez MT, et al. Indications for active case deficiency detection in a Portuguese population. COPD. 2018;15 searches and intravenous alpha-1 antitrypsin treatment for patients (1):4–9. doi:10.1080/15412555.2017.1414779 with alpha-1 antitrypsin deficiency chronic pulmonary obstructive 26. Greulich T, Ottaviani S, Bals R, et al. Alpha1-antitrypsin deficiency - disease: an update. Arch Bronconeumol. 2015;51(4):185–192. diagnostic testing and disease awareness in Germany and Italy. doi:10.1016/j.arbres.2014.05.008 Respir Med. 2013;107:1400–1408. 9. Blanco I, Bueno P, Diego I, et al. Alpha-1 antitrypsin Pi*Z gene 27. Taliercio RM, Chatburn RL, Stoller JK. Knowledge of alpha-1 anti­ frequency and Pi*ZZ genotype numbers worldwide: an update. trypsin deficiency among internal medicine house officers and Int J Chron Obstruct Pulmon Dis. 2017;12:561–569. doi:10.2147/ respiratory therapists: results of a survey. Respir Care. 2010;55 COPD.S125389 (3):322–327. 2430 submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 DovePress Dovepress Belmonte et al 28. Esquinas C, Barrecheguren M, Sucena M, Rodríguez E, Fernandez S, 34. Lopez-Campos JL, Casas-Maldonado F, Torres-Duran M, et al. Miravitlles M. Practice and knowledge about diagnosis and treatment Results of a diagnostic procedure based on multiplex technology on of alpha-1 antitrypsin deficiency in Spain and Portugal. BMC Pulm dried blood spots and buccal swabs for subjects with suspected Med. 2016;16:64. doi:10.1186/s12890-016-0222-4 alpha1 antitrypsin deficiency. Arch Bronconeumol. 2020;14:S0300– 29. de la Roza C, Rodriguez-Frias F, Lara B, Vidal R, Jardi R, 2896(20)30131–9. doi:10.1016/j.arbres.2020.04.014 Miravitlles M. Results of a case-detection programme for 35. Barrecheguren M, Torres-Duran M, Casas-Maldonado F, alpha1-antitrypsin deficiency in COPD patients. Eur Respir J. Miravitlles M. Spanish implementation of the new international 2005;26:616–622. doi:10.1183/09031936.05.00007305 alpha-1 antitrypsin deficiency international registry: the European 30. Greulich T, Rodríguez-Frias F, Belmonte I, Klemmer A, Alpha-1 Research Collaboration (EARCO). Arch Bronconeumol. Vogelmeier CF, Miravitlles M. Real world evaluation of a novel 2020;pii:S0300–2896(20)30061–2. doi:10.1016/j.arbres.2020.02.003 lateral flow assay (AlphaKit® QuickScreen) for the detection of 36. Sanders CL, Ponte A, Kueppers F. The effects of inflammation on alpha-1-antitrypsin deficiency. Respir Res. 2018;19:151. alpha 1 antitrypsin levels in a National Screening Cohort. COPD. doi:10.1186/s12931-018-0826-8 2018;15:10–16. doi:10.1080/15412555.2017.1401600 31. Jain A, McCarthy K, Xu M, et al. Impact of a clinical decision 37. Janciauskiene S, DeLuca DS, Barrecheguren M, Welte T, support system in an electronic health record to enhance detection Miravitlles M. Serum levels of alpha1-antitrypsin and their relation­ of alpha 1 antitrypsin deficiency. Chest. 2011;140(1):198–204. ship with COPD in the general Spanish population. Arch doi:10.1378/chest.10-1658 Bronconeumol. 2020;56:76–83. doi:10.1016/j.arbres.2019.03.001 32. Molina J, Flor X, Garcia R, Timiraos R, Tirado-Conde G, 38. Ellis P, Turner A. What do alpha-1 antitrypsin levels tell us about Miravitlles M. The IDDEA project: a strategy for the detection of chronic inflammation in COPD? Arch Bronconeumol. 2020;56 alpha-1 antitrypsin deficiency in COPD patients in the primary care (2):72–73. doi:10.1016/j.arbres.2019.06.010 setting. Ther Adv Respir Dis. 2011;5:237–243. doi:10.1177/ 39. Kueppers F. PredictAAT: accounting for inflammation in the diag­ 1753465811404919 nosis of alpha 1 antitrypsin deficiency. COPD. 2020;1–4. 33. Rodriguez-Frias F, Miravitlles M, Vidal R, Camos S, Jardi R. Rare doi:10.1080/15412555.2020.1812055 alpha-1-antitrypsin variants: are they really so rare? Ther Adv Respir Dis. 2012;6(2):79–85. doi:10.1177/1753465811434320 International Journal of Chronic Obstructive Pulmonary Disease Dovepress Publish your work in this journal The International Journal of COPD is an international, peer-reviewed protocols. This journal is indexed on PubMed Central, MedLine journal of therapeutics and pharmacology focusing on concise rapid and CAS. The manuscript management system is completely online reporting of clinical studies and reviews in COPD. Special focus is and includes a very quick and fair peer-review system, which is given to the pathophysiological processes underlying the disease, inter­ all easy to use. Visit http://www.dovepress.com/testimonials.php to vention programs, patient focused education, and self management read real quotes from published authors. Submit your manuscript here: https://www.dovepress.com/international-journal-of-chronic-obstructive-pulmonary-disease-journal submit your manuscript | www.dovepress.com International Journal of Chronic Obstructive Pulmonary Disease 2020:15 2431 DovePress