Nanopia kl 6 assay

The Nanopia KL-6 assay (SEKISUI MEDICAL, Tokyo, Japan) was used to measure blood KL-6 levels. All serum samples were immediately transported to the central lab of each hospital and centrifuged after blood collection. The latex-enhanced immunoturbidimetric assay, which measures changes in absorbance by agglutination, was used to measure KL-6 concentration.

Blood samples were obtained by venipuncture and stored at -80°C until measurement. Plasma KL-6 levels were measured by a Nanopia KL-6 assay (SEKISUI MEDICAL, Tokyo) using a latex-enhanced immunoturbidimetric assay method as used in previous studies [30 (link),31 (link)]. Previous study conducted in our institution reported that within-laboratory precisions of Nanopia KL-6 assay were < 2% of coefficient of variation [32 (link)].

As biomarkers, serum KL-6 and monocyte count were evaluated in this study. The serum KL-6 concentration was measured using a Nanopia KL-6 assay (SEKISUI MEDICAL, Tokyo) using a latex-enhanced immunoturbidimetric assay method. The monocyte count was calculated using white blood cell differential on CBC. Serum KL-6 ≥ 1000 U/mL is well known to predict poor prognosis in IPF, and, as such, was set as the cut-off value in this study^{15 (link),17 (link),26 (link)}. However, the optimal cut-off value of monocytes has not yet been validated. Therefore, we arbitrarily set monocytes ≥ 600/μL as a cut-off value based on recent studies^{20 (link),21 (link)}. Moreover, to determine whether the interval change in KL-6 level is related to DP, we divided patients into two groups according to the KL-6 level at the 3rd month from the baseline. Patients who had increased KL-6 levels at the 3rd month compared to baseline level were classified as the increased KL-6 group, and other patients were classified as the non-increased KL-6 group.

Serum samples were obtained by venipuncture at time of first evaluation and were stored at −80 °C until analysis. Serum KL-6 was measured by NANOPIA® KL-6 assay (SEKISUI Diagnostics, UK; upper limit of normal <458 U/mL as determined in 142 Caucasian healthy subjects). GM-CSF autoantibody (Abs) concentration was measured according to Kitamura, T., et al. [28 (link)]. Recombinant GM-CSF (Sargramostim, Genzyme, Cambridge, USA) was used to coat plates, as standard we used monoclonal human-anti-human GM-CSF antibody (BI01049904) provided by Boehringer Ingelheim, Germany. The detection limit of this assay is 0.2 μg/mL. GM-CSF Abs values <3 μg/mL are considered normal, 3–7 μg/mL intermediate, and >7 compatible with autoimmune PAP, according to Inoue et al. 2008 [4 (link)]. LDH was routinely measured in serum (normal value for LDH in our laboratory < 225 IU/L).

Demographic, clinical, and laboratory information were obtained through medical chart review. PFT results included percent diffusion capacity for carbon monoxide (DLCO%), forced vital capacity (FVC%), and forced expiratory volume-one second (FEV1%). Inflammatory markers such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were recorded to the nearest date of serum collection, not exceeding 30 days. Mean and highest ESR and CRP values were calculated between the first two chest HRCTs. Rheumatoid factor (RF) was measured with immunoturbidimetric assay (reference range < 15 IU/mL), and anti-cyclic citrullinated peptide (anti-CCP) was measured with chemiluminescent microparticle immunoassay (reference range, <5.0 IU/mL). Serum level of Kerbs von den Lungen 6 (KL-6) was measured using Nanopia KL-6 assay (SEKISUI MEDICAL CO., LTD., Tokyo, Japan).