Validation and development of an immunonephelometric assay for the determination of alpha-1 antitrypsin levels in dried blood spots from patients with COPD

Abstract

OBJECTIVE: To validate and develop an immunonephelometric assay for the determination of alpha-1 antitrypsin (AAT) levels in dried blood spots from COPD patients in Brazil. METHODS: We determined AAT levels in serum samples and dried blood spots from 192 COPD patients. For the preparation of dried blood spots, a disk (diameter, 6 mm) was placed into a tube, eluted with 200 µL of PBS, and stored overnight at 4ºC. All of the samples were analyzed by immunonephelometry in duplicate. We used the bootstrap resampling method in order to determine a cut-off point for AAT levels in dried blood spots. RESULTS: The correlation coefficient between the AAT levels in serum samples and those in dried blood spots was r = 0.45. For dried blood spots, the cut-off value was 2.02 mg/dL (97% CI: 1.45-2.64 mg/dL), with a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 95.7%, 27.2%, and 100%, respectively. CONCLUSIONS: This method for the determination of AAT levels in dried blood spots appears to be a reliable screening tool for patients with AAT deficiency.

Prevalence of alpha-1 antitrypsin deficiency and allele frequency in patients with COPD in Brazil

“…The decision to use the maximum confidence interval of the cut-off point for AAT values in eluates 17 was due to the need to identify all individuals who were suspected of having AATD and should therefore undergo genotyping, thus minimizing the chances of not identifying patients with AATD. Although AATD is one of the most common genetic diseases, the prevalence of AATD is low.…”

“…However, failure to identify any patient with AATD would have had an impact on the final results of the present study. Nevertheless, the use of the maximum confidence interval width resulted in more patients being re-evaluated 17 . The cut-off point of 113 mg/dL was used in an attempt to identify not only patients with severe AATD but also those with moderate AATD 20 …”

“…In the first phase, all patients underwent determination of AAT levels in dried blood spot (DBS) samples in order to identify those with a possible diagnosis of AATD. In the second phase, patients with DBS AAT levels ≤ 2.64 mg/dL (suspected AATD) underwent determination of serum AAT levels 17 . Finally, in the third phase, patients with serum AAT levels of < 113 mg/dL underwent genotyping.…”

“…To determine the sensitivity and specificity of the eluate method, Zillmer et al used the bootstrap resampling method, comparing the AAT values measured in serum with those measured in eluates from DBS samples in order to determine a cut-off point for AAT values in eluates; the value obtained was 2.02 mg/dL (97% CI: 1.45-2.64). 17 All patients whose DBS AAT levels were below 2.64 mg/dL underwent measurement of serum AAT levels in order to prevent AATD from going undiagnosed. …”

Diagnosis and treatment of pulmonarydisease in α1-antitrypsin deficiency: a statement of European Respiratory Society

“…В последнее десятилетие широкое распространение получили наиболее воспроизводимые и надежные количественные методы, основанные на применении высушенных образцов крови, которые по зволяют централизовать тестирование больных [27][28][29].…”