Behring nephelometer 2

IgG4 levels in serum (mg/dL) were measured in 23 patients who underwent steroid therapy using automated nephelometry^{[11 (link)]} (Behring Nephelometer II; Dade Behring, Inc., Newark, DE).

All blood samples were collected during the midweek dialysis from the AV fistula, immediately after the insertion of the dialysis cannula but before the administration of heparin. Blood was sampled in 4 c.c. Venoject II tubes and centrifuged (10 min, 3000 rpm) and stored at −70°C pending analyses, if not analyzed immediately. Serum albumin, urea, creatinine (Cr), total cholesterol, and triglyceride (TG) were determined according to standard methods. The serum levels of high-sensitivity C-reactive protein (hsCRP) were measured using a Behring Nephelometer II (Dade Behring, Tokyo, Japan).

Venous blood was extracted into Vacutainer® tubes and centrifuged at 1500 g for 10 min at 4 °C, after which biochemical parameters were evaluated as above mentioned^{24 (link)}. To quantify triglycerides, glucose and total cholesterol levels in serum, was employed an enzymatic method. Insulin levels were measured by immunochemiluminescence and homeostasis model assessment (HOMA-IR = [fasting insulin (μU/ml) x fasting glucose (mg/dl)]/405) was used to calculate insulin resistance. An automatic glycohemoglobin analyser (Arkray Inc., Kyoto, Japan) was used to determine the percentage of HbA1c. A Beckman LX-20 autoanalyzer (Beckman Coulter, La Brea, CA, US) was employed to assesed high density lipoprotein (HDL) levels, low density lipoprotein (LDL) content was estimated with Friedewald’s formula, and high-sensitive C-reactive protein (hs-CRP) levels were measured by an immunonephelometric assay (Behring Nephelometer II, Dade Behring, Inc., Newark, DE, USA).

Plasma AST, ALT, CK, TC, TG and HDL-C concentrations were determined by standard routine enzymatic methods. LDL-C concentrations were calculated with the Friedewald formula. Non-HDL-C was calculated by subtracting HDL-C from TC, whereas REM-C was calculated by subtracting HDL-C and LDL-C from TC. ApoA-I, apoB, apoC-III and hsCRP concentrations were determined by immunoassay. Lp(a) was measured by immunonephelometric assay (Behring Nephelometer II; Dade Behring Inc, Milton Keynes, UK.). The treatment effect of LA in 6 HoFH patients prior to evinacumab were expressed as time-average LDL-C using Kroon’s equation (Cmean = Cmin + K(Cmax − Cmin), where K is the rebound coefficient for HoFH [28 (link),29 (link)].

After fasting for 12 hours, serum or plasma samples were obtained and the patients underwent the following laboratory blood analysis: glucose and uric acid (UA) were evaluated by a biochemical autoanalyzer (Dimension Dade AR, Dade Behring®, Deerfield, IL, USA), using Dade Behring kits; plasma insulin level and anticyclic citrullinated peptide (anti-CCP) antibody were determined by chemiluminescence microparticle immunoassay (Architect, Abbott Laboratory, Abbott Park, IL, USA). The homeostasis model assessment-IR (HOMA-IR) was used as a surrogate measurement of insulin resistance [28 (link)]. Consider the following: HOMA-IR = insulin fasting (μU/mL) × glucose fasting (nmol/L)/22.5. IR was considered when HOMA-IR ≥ 2.114 [8 (link)]. Serum high sensitivity CRP (hsCRP) and rheumatoid factor (RF) were measured using a nephelometric assay (Behring Nephelometer II, Dade Behring, Marburg, Germany). TNF-α levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using a commercial immunoassay ELISA (Ready-SET-Go! Set, e-Bioscience, San Diego, California, USA). Erythrocyte sedimentation rate (ESR) was obtained by automated kinetic-photometric method (Ves-Matic CUBE 30, DIESSE, Siena, Italy).