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Clinical Chemistry 52: 899-901, 2006; 10.1373/clinchem.2005.062059
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(Clinical Chemistry. 2006;52:899-901.)
© 2006 American Association for Clinical Chemistry, Inc.

Letters to the Editor

Validation of a Rapid, Simple Method to Measure {alpha}1-Antitrypsin in Human Dried Blood Spots

Marina Gorrini1, Ilaria Ferrarotti1, Anna Lupi2, Tiziana Bosoni3, Paola Mazzola1, Roberta Scabini1, Ilaria Campo1, Michele Zorzetto1, Francesco Novazi3 and Maurizio Luisetti1,a

1 Centro Diagnosi Deficit, Ereditario AAT, Laboratorio di Biochimica, e Genetica, Clinica Malattie Apparato, Respiratorio, and, 3 Servizio Analisi Chimico Cliniche, Istituto di Ricovero e Cura, a Carattere Scientifico, Policlinico San Matteo, Pavia, Italy
2 Dipartimento di, Biochimica A. Castellani, Università di Pavia, Pavia, Italy

aAddress correspondence to this author at: Clinica Malattie Apparato Respiratorio, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Policlinico San Matteo, Piazza Golgi, 27100 Pavia, Italy. Fax 39-0382-502269; e-mail m.luisetti{at}smatteo.pv.it.


To the Editor:

Screening for {alpha}1-antitrypsin deficiency (AATD) can be performed with dried blood spots (DBS) on filter paper, which allows quantification of AAT by immunonephelometry (1), identification of the AAT phenotype by isoelectric focusing(2)(3), and/or AAT genotyping (4). In most diagnostic flow-charts, important decisions often depend on the AAT concentration determined from DBS; however, current methods are imprecise, and results from DBS are not identical to those obtained using serum samples(4). We wished to develop a method for quantifying AAT in DBS.

We collected 75 specimens from healthy blood donors and 74 from patients with suspected AATD. Samples were centrifuged for 8 min at 1620g, and the serum or plasma, without manifest hemolysis, was separated and stored at –80 °C. Blood was spotted on filter paper with dashed-line 13-mm printed circles (Schleicher & Schuell Grade 903; lot. W-011) and air-dried. Cards stored in plastic bags at room temperature can be kept for at least 1 year. We measured AAT in serum or plasma by nephelometry (Array 360 System) with a polyclonal anti-human AAT goat antibody and calibrator (CAL2, assigned AAT value of 2.05 g/L), all from Beckman Coulter. We assayed Level 1 and 3 Liquichek Immunology Controls (Bio-Rad Laboratories) daily. Samples were automatically diluted 1 to 216 (AAT range, 0.06–0.60 g/L). If necessary, a 1:36 dilution was prepared. Both plasma and serum samples were available for 50 patients: we found that plasma and sera were interchangeable (not shown). The reference interval for AAT in serum or plasma assayed in our laboratory was 0.83–1.99 g/L.

We calculated that at an average hematocrit of 45%, with average absorption of 22.2 µL of blood by each 6-mm paper disk (the size obtained from the filter circle by use of a DBS Puncher from Perkin-Elmer), each DBS sample contains 12.2 µL of plasma or serum. To elute 1 disk, a volume of 266 µL of water is needed for overnight elution at room temperature; thus, a theoretical dilution of 1 to 21.8 was applied to the blood samples. We included a daily in-house quality control with DBS prepared with CAL2 in EDTA-blood from a patient without detectable AAT (nominal values, 0.10–3.00 g/L). Eluted DBS samples for the nephelometric assay were not diluted (AAT reading range, 0.003–0.028 g/L) or were diluted 1 to 6 (AAT reading range, 0.028–0.167 g/L) to be in the working range of the assay.

We evaluated the effect of different matrices on the recovery of AAT, defined as the percentage of AAT in the DBS compared with the corresponding serum or plasma concentration (see Fig. 1 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue5). The recovery of AAT in the eluate from DBS at a 1:21.8 dilution varied according to the complexity of the matrix, which acts as a molecular sieve, and the concentration of the protein itself. Thus, we used 2 regression curves to evaluate all serum/plasma samples vs the corresponding DBS (Fig. 1 ). The choice of curve was based on the nephelometric reading of the eluate. If the DBS reading, increased 21.8-fold, was <0.22 g/L, curve A (range, 0.16–0.53 g/L) was used, whereas if the DBS reading was ≥0.22 g/L, curve B (range, 0.54–2.93 g/L) was used. The correlation coefficients were 0.9889 and 0.9098 for samples with low and medium-to-high concentrations of AAT, respectively.


Figure 1
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  		Figure 1. Regression results for DBS eluate vs serum or plasma samples in a serum/plasma range of 0.14–0.53 g/L (A) and a serum/plasma range of 0.54–3.16 g/L (B).

(A), this regression shows 17 samples with very low AAT concentrations (0.16–0.21 g/L) with 100% protein recovery from the DBS, and 10 samples with low concentrations (0.22–0.53 g/L) with 55% protein recovery [regressed values (95% confidence interval), 0.21–0.30 g/L]. Equation for the line: AAT (g/L) = 0.7907x + 0.0410 g/L (r2 = 0.9889). (B), this regression shows 122 samples with medium or high AAT concentrations (0.54–2.93 g/L), with 55% to 30% recovery [regressed values (95% confidence interval), 1.28–1.44]. Equation for the line: AAT (g/L) = 2.7087x + 0.0101 g/L (r2 = 0.9098).

We evaluated the precision, recovery, and sensitivity of the AAT assay for the concentration range read by our instrument (0.03–0.17 g/L) by adding calibrator to the DBS, prepared with CAL2 (successive additions of 0.03 g/L) in EDTA-blood from a patient without detectable AAT. The results are presented in Table 1 of the online Data Supplement. Intra- and interday imprecision (CV) was 0.6%–3.2% and 0.3%–3%, respectively (see Table 1 in the online Data Supplement).

We studied 114 samples submitted to our center for a laboratory diagnosis of AATD. These samples had been genotyped and sequenced (5)(6). Fifty-one samples came from PI*MM individuals (healthy), and 63 came from patients with AATD. The AATD was either severe or intermediate: 8 were PI*MR (where R indicates rare deficient variants)(6), 8 were PI*MS, 25 were PI*MZ, 4 were PI*SZ, 14 were PI*ZZ, and 4 were PI*ZR. In this group, albeit small, an AAT concentration of 1.13 g/L represented the best cutoff to differentiate AATD patients from healthy individuals (sensitivity, 0.92; specificity, 0.90).

In conclusion, this method allowed AAT to be assayed from a 6-mm disk and provided the same quantitative results on both conventional and DBS samples.


Acknowledgments

We gratefully acknowledge the skillful technical assistance of Carla Villani and Laura Pirolini. This research was supported by a Ricerca Corrente from the Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Matteo, Pavia; a grant from Istituto Superiore di Sanità Progetto "Malattie Rare"; and an educational grant from Bayer.


References

  1. Rodriguez F, Jardì R, Costa X, Cotrina M, Galimany R, Vidal R, et al. Rapid screening for {alpha}1-antitrypsin deficiency in patients with chronic obstructive pulmonary disease using dried blood specimens. Am J Respir Crit Care Med 2002;166:814-817.[Abstract/Free Full Text]
  2. Wencker M, Marx A, Konietzko N, Schaefer B, Campbell EJ. Screening for {alpha}1-Pi deficiency in patients with lung disease. Eur Respir J 2002;20:319-324.[Abstract/Free Full Text]
  3. Spence WC, Morris JE, Pass K, Murphy PD. Molecular confirmation of {alpha}1-antitrypsin genotypes in newborn dried blood specimens. Biochem Med Metab Biol 1993;50:233-240.[CrossRef][Medline] [Order article via Infotrieve]
  4. Costa X, Jardi R, Rodriguez F, Miravittles M, Cotrina M, Gonzalez C, et al. Simple method for {alpha}1-antitrypsin deficiency screening by use of dried blood spot specimens. Eur Respir J 2000;15:1111-1115.[Abstract]
  5. Ferrarotti I, Zorzetto M, Scabini R, Mazzola P, Campo I, Luisetti M. A novel method for rapid genotypic identification of {alpha}1-antitrypsin variants. Diagn Mol Pathol 2004;13:160-163.[CrossRef][Medline] [Order article via Infotrieve]
  6. Ferrarotti I, Baccheschi J, Zorzetto M, Tinelli C, Corda L, Balbi B, et al. Prevalence and phenotype of subjects carrying rare variants in the Italian Registry for {alpha}1-antitrypsin deficiency. J Med Genet 2005;42:282-287.[Free Full Text]



The following articles in journals at HighWire Press have cited this article:


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M. Zorzetto, E. Russi, O. Senn, M. Imboden, I. Ferrarotti, C. Tinelli, I. Campo, S. Ottaviani, R. Scabini, A. von Eckardstein, et al.
SERPINA1 Gene Variants in Individuals from the General Population with Reduced {alpha}1-Antitrypsin Concentrations
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[Abstract] [Full Text] [PDF]

This Article
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