A dried blood spot test for diagnosis of autoimmune pulmonary alveolar proteinosis

Abstract

Granulocyte/macrophage colony-stimulating factor autoantibodies (GMAbs) mediate the pathogenesis of autoimmune pulmonary alveolar proteinosis (autoimmune PAP) and their quantification in serum by enzyme-linked immunosorbent assay (ELISA) - the serum GMAb test - is the 'gold standard' for diagnosis of autoimmune PAP. Because GMAbs are high in autoimmune PAP and low or undetectable in healthy people, we hypothesized that the ELISA could be adapted for evaluation of blood obtained from the fingertip using a dried blood spot card (DBSC) for specimen collection. Here, we report development of such a method - the DBSC GMAb test - and evaluate its ability to measure GMAb concentration in blood and to diagnose autoimmune PAP. Fresh, heparinized whole blood was obtained from 60 autoimmune PAP patients and 19 healthy people and used to measure the GMAb concentration in blood (by the DBSC GMAb test). After optimization, the DBSC GMAb test was evaluated for accuracy, precision, reliability, sensitivity, specificity, and ruggedness. The coefficient of variation among repeated measurements was low with regard to well-to-well, plate-to-plate, day-to-day, and inter-operator variation, and results were unaffected by exposure of prepared DBSC specimens to a wide range of temperatures (from -80 °C to 65 °C), repeated freeze-thaw cycles, or storage for up to 2.5 months before testing. The limit of blank (LoB), limit of detection (LoD), and lower limit of quantification (LLoQ), were 0.01, 0.21, and 3.5 μg/ml of GMAb in the blood, respectively. Receiver operating curve characteristic analysis identified 2.7 μg/ml as the optimal GMAb concentration cutoff value to distinguish autoimmune PAP from healthy people. This cutoff value was less than the LLoQ and the ranges of GMAb results for autoimmune PAP patients and healthy people were widely separated (median (interquartile range): 22.6 (13.3-43.8) and 0.23 (0.20-0.30) μg/ml, respectively). Consequently, the LLoQ is recommended as the lower limit of the range indicating a positive test result (i.e., that autoimmune PAP is present); lower values indicate a negative test result (i.e., autoimmune PAP is not present). Among the 30 autoimmune PAP patients and 19 healthy people evaluated, the sensitivity and specificity of the DBSC GMAb test were both 100% for a diagnosis of autoimmune PAP. Results demonstrate the DBSC GMAb test reliably measures GMAbs in blood and performs well in the diagnosis of autoimmune PAP.

Keywords: Autoantibodies; Autoimmune PAP; Diagnosis; Enzyme-linked immunosorbent assay; Granulocyte/macrophage-colony stimulating factor.