Elsevier

Translational Research

Volume 150, Issue 5, November 2007, Pages 267-274
Translational Research

Review article
Laboratory diagnosis of alpha1-antitrypsin deficiency

https://doi.org/10.1016/j.trsl.2007.08.001Get rights and content

The laboratory diagnosis of alpha1-antitrypsin (AAT) deficiency (AATD) has evolved over the last 40 years since the first cases of the disorder were reported. It is currently performed in specialized centers, and it requires a combination of different biochemical methods: nephelometric AAT concentration, isoelectric focusing, genotyping, and sequencing. The availability of matrices such as the dried blood spot have facilitated the implementation of laboratory analyses for AATD, but they have also challenged laboratories to develop more reliable and reproducible techniques starting from dried blood. In this article, we describe the protocols we have optimized for AATD diagnosis from dried blood spot, in an attempt to hopefully provide useful information for physicians and scientists involved in this diagnostic line. We also describe the diagnostic flowchart for AATD detection that we have developed accordingly.

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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    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.).

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