Review articleLaboratory diagnosis of alpha1-antitrypsin deficiency
Section snippets
Biochemical methods to diagnose AATD
The above reports clearly imply that the laboratory diagnosis of AATD is not just a matter of degree, although the AAT serum level is the most important determinant of the risk for lung damage.11 The precise determination of the deficiency alleles carried by an AATD individual may help to evaluate the risk for the liver disease: Null alleles do not indicate a risk for liver pathology, whereas the identification of alleles associated with a high degree of AAT polymerization, such as Z or Mmalton,
Handling of DBS
Most AATD detection/screening programs currently use DBS, and the filter papers (903; Schleicher & Schuell, Dassel, Germany) carry from 3 to 6 dashed-line, 13-mm printed circles. Fresh blood collected by venotip or fingertip puncture should totally fill the circles as well as the reverse side of the filter. The filter should be allowed to air dry for at least 12 h and then should be shipped. DBS filters are provided with a specific bar code, and medical staff fill in a form with information on
Quantitative determination
The nephelometric determination of AAT levels in DBS samples is performed with an Array 360 System (Beckmann Coulter S.P.A, Milan, Italy), using a polyclonal anti-human AAT goat antibody. A calibrator (CAL2, Beckmann Coulter) with an assigned AAT value is used as a standard. To monitor the precision of the testing procedures, a Level 1 and 3 Liquichek Immunology Control (Bio-Rad Laboratories, Irvine, Calif) is performed daily. To make precise blood spot punches, a Wallac 1296-071 DBS Puncher
Sequence analysis
Sequence analysis, which is required in case of discrepancies between AAT nephelometric level and genotyping, is performed on CEQ 8800 Genetic Analysis System (Beckman Coulter).
Polymerase chain reaction (PCR) reaction conditions are as follows: 1 μL of each primer and 0.5 μL of AccuPrimeTM Taq High Fidelity (Bioline, London, United Kingdom) are added to 100 ng of DNA in a 25-μL final volume of a solution containing 600-mmol/L Tris-SO4, 180-mmol/L (NH4)2SO4, 2 mmol/L each dNTPs, Thermostable
Conclusions
The laboratory methodologies for the diagnosis of AATD have evolved slowly over the last 40 years, since the first cases of the disorder were reported. Currently in specialized centers it requires a combination of different biochemical methods. The availability of matrices such as DBS has facilitated the laboratory diagnosis of AATD, but it has also challenged laboratories to develop more reliable and reproducible techniques starting from dried blood. Undoubtedly, the evolution of technologies
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Non-invasive diagnosis and follow-up of rare genetic liver diseases
2022, Clinics and Research in Hepatology and GastroenterologyCitation Excerpt :Despite the establishment of this technique as a reference method for the exploration of alpha-1 antitrypsin, complementary means of analysis provided by molecular biology techniques are being increasingly used in the diagnosis of alpha-1 antitrypsin deficiency. The first molecular approach is investigation of the two deficient alleles, PiS and PiZ, which are most commonly reported [23]. However, deficient cases lacking mutations in the exons coding for alpha-1 antitrypsin have been reported.
Investigating the Link between Alpha-1 Antitrypsin Deficiency and Abdominal Aortic Aneurysms
2021, Annals of Vascular SurgeryCitation Excerpt :Amplification by PCR has been performed with the following primers: - P3M (5¹CTTCCAAACCTTCACCTGGT3¹) and P3P (5¹GTCCTCATGAGCATGACGGCG3¹) for amplification of exon III S-variant; -5M (5¹GAGCCCCTTGCTCGCCTGGATC3¹) and 5P (5¹CAGGAAACATGAGGAGGATGATTTAC3¹) to amplify and search the exon V variant Z.45 The sequencing technique was necessary when a discrepancy between the results obtained with the AAT assay and the genotyping occurred.
Quantification of circulating alpha-1-antitrypsin polymers associated with different SERPINA1 genotypes
2024, Clinical Chemistry and Laboratory MedicineMeasuring of Alpha-1 Antitrypsin Concentration by Nephelometry or Turbidimetry
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Supported in part by the Fondazione IRCCS Policlinico San Matteo Ricerca Corrente grant, the Istituto Superiore di Sanitá programma di Collaborazione Italia-USA 2005 N.526/A4, an educational grant from Bayer HealthCare, and from the European Laurell’s disease training awards for 2006 (to I.F.).