Chemiluminescence immunoassay

Using sterile venipuncture, six milliliters of fresh venous blood were taken. Two milliliters of blood were immediately transferred to an EDTA vacutainer tube for the total RNA extraction and complete blood count (CBC) via the Sysmex XN-1000 Automated Hematology Analyzer, Kobe, Japan; 2 mL was transferred to a sodium citrate vacutainer tube for the D-dimer test by the Fully Automated Coagulation System, Diagnostic Stago, France. The remaining 2 mL was transferred to a plain tube and centrifuged at 4000 rpm for 20 min. The serum was then refrigerated at −20 °C until the measurements of serum ferritin using the Abbott chemiluminescence immunoassay, North Chicago, IL, USA; of the C-reactive protein (CRP) using the nephelometric method by Mispa-i2, and of interleukin 6 (IL-6) using the enzyme-linked immunosorbent assay (ELISA) human kits.

A serum thyroid stimulating hormone (TSH) level of higher than 30 μIU/mL was achieved by thyroid hormone withdrawal (THW) before RIT. All patients were instructed with a low iodine diet from the beginning of THW till 3 weeks after RIT. Patients were administrated with a radioactive iodine dose of 3.7–7.4GBq (100-200mCi) according to the Chinese guideline (5 (link)). The second RIT was performed about 6 months after first therapy (5 (link)). All patients were under TSH suppressive therapy by using sodium levothyroxine with TSH <0.1 μIU/mL.
The functional detection sensitivities of Tg and TgAb by electrochemiluminescence immunoassay (Abbott Laboratories, Chicago, IL, USA) were 0.2 ng/mL and 20 IU/mL. TSH was determined by chemiluminescence immunoassay (Abbott Laboratories, Chicago, IL, USA), with a measuring range from 0.35 to 4.94 μIU/mL. A serum stimulated or suppressed Tg, TSH, TgAb were usually followed 1 day before RIT and 2–3 months after RIT.
¹³¹I-WBS was obtained by using Discovery NM/CT 670 (General Electric Medical Systems, Milwaukee, Wisconsin, USA) equipped with high-energy parallel-hole collimators, and a 20% energy window was centered at 364 keV, at a table speed of 14 cm/min for a total time of 20 min (256×1024 matrix). Post-therapeutic whole-body scan (Rx-WBS) was performed 3-6 days after RIT (5 (link)).

The analyses of efficacy included all patients who could be followed for 24 weeks after the completion of the 48-week PEG-IFN treatment. Blood samples were taken at 4-week intervals during the treatment and once at 24 weeks after the completion of treatment. The primary outcome measure was HBeAg seroconversion (defined by the loss of HBeAg and presence of anti-HBe in serum) 24 weeks after the completion of the 48-week treatment, which was defined as a treatment response. The secondary outcome measures were as follows: loss of HBeAg, loss of HBsAg, HBsAg seroconversion (defined as the loss of HBsAg and the presence of anti-HBs in serum), undetectable HBV DNA, and normalization of ALT levels 24 weeks after the completion of the 48-week treatment. Serum HBeAg, anti-HBe, and anti-HBs levels were measured by chemiluminescence immunoassay (Abbott Laboratories, Chicago, IL). Serum HBs levels were measured by using an ARCHITECT HBsQT assay kit (Abbott Laboratories), which has a lower limitation of detection of 0.05 IU/mL. Serum HBV DNA was quantified with the COBAS TaqMan HBV DNA test (Roche Diagnostics, Basel, Switzerland), which has a dynamic range of 2.1–9.0 log copies/mL. The conversion factor between HBV copies/mL and HBV IU/mL is considered to be 5.82 copies/IU. Genotyping of HBV was determined by the polymerase chain reaction (PCR)-Invader assay (12 (link)).

Serum PTH was evaluated using the chemiluminescent microparticle immunoassay method through the ARCHITECT ci 4100 equipment (Abbott Diagnostics, Abbott Park, IL, USA), with reference values (RVs) of 15.0–68.3 pg/mL; the intra-assay and inter-assay coefficients of variation were 6.1 and 6.4%, respectively. Total serum calcium was evaluated using the NM-BAPTA method and corrected via albumin levels (corrected calcium = 0.8 × (4.0−serum albumin) + serum calcium) with RVs of 8.6 to 10.0 mg/dL. Serum phosphorus was evaluated using the Molybdate UV and colorimetric (xylidil blue) methods, with RVs of 2.5–4.5 mg/dL. The electrolyte tests were conducted using Cobas 8000 c702 equipment (Roche Diagnostics, Indianapolis, IN, USA). Free thyroxine and TSH were evaluated using a chemiluminescence immunoassay (Abbot, Chicago, IL, USA), with RVs of 0.7–1.48 ng/mL and 0.35–4.94 mUI/L, respectively. These tests were all conducted in the HCPA central laboratory.

All blood samples were detected in a College of American Pathologists (NO. 7217913)-accredited laboratory. We performed a 1mg overnight dexamethasone suppression test (DST) by administering 1mg of dexamethasone at 24:00, with measurement of cortisol the following morning (between 08:00 and 09:00 hours). Serum cortisol and 24h-UFC were measured by using an Access Immunoassay System (A Beckman Coulter Corp, Fullerton, CA, USA). Plasma ACTH levels were measured by using an ELSA-ACTH immunoradiometric assay method (Cisbio Bioassays, Codolet, France). The intraassay and interassay coefficients of variation were 6.1% and 5.3% for ACTH. The normal range for ACTH is 12-78 pg/ml. Serum-free testosterone (T) and androstenedione (AD) were measured by radioimmunoassay. Other sex hormones, such as dehydroepiandrosterone sulfate (DHEAS), SHBG, and T were measured by using a chemiluminescence immunoassay (Abbott Laboratories, Abbott Park, IL).