Rapid intraoperative method for the identification of metastatic lymph nodes from thyroid carcinoma
Longshun Shiad, Minhao Xiead(Co-first authors), Guo Wub, Jun Fanad, Mingming Guoad, Runlin Yangad, Jue Zhangad, Yi Zhangad, Bin Zhouad, Zhongwei Lvc (Co-corresponding authors)
In this study, we established a rapid method for intraoperative identification of thyroid carcinoma metastatic lymph nodes. We developed a rapid method of thyroid globulin (Tg) detection called Tg-POCT based on time-resolved fluorescence immunoassay and validated reagent performance. Thyroid and lymph node tissues with metastatic thyroid cancer were considered as the positive group, and muscle, fiber, fat, and other tissues were considered as the negative control group to verify the above method combined with fine needle puncture. CV of the same batch number was 8.38% and that of different batch numbers was 11.24%. The minimum detection limit was 0.02 ng/mL. The test conformity of tissue samples was 100% by using the self-designed reference value. Thus, Tg-POCT is feasible as a rapid intraoperative method to identify thyroid cancer metastatic lymph nodes.
Introduction
In recent years, we attempted to determine thyroglobulin (Tg) content by fine needle aspiration (FNA) eluent, which showed positive significance in the qualitative analysis of thyroid tumor-related cervical lymph nodes [1-2]. Tg is a macromolecule glycoprotein, synthesized only by thyroid epithelial cells, with a relative molecular weight of 660 KD, and its presence can indicate the existence of thyroid follicular epithelial cells. In addition to the high levels content of Tg in normal thyroid tissues, high levels of Tg are observed in well-differentiated thyroid cancer tissues and lymph node tissues with thyroid cancer metastasis [3].Therefore, Tg detection by puncture cell eluent can be used as a supplement to FNA to improve accuracy in identifying the metastatic lymph nodes of thyroid cancer. In addition, the rapid detection of Tg in puncture cell eluent during surgery will significantly improve the quality of thyroid cancer surgery and reduce the risk of postoperative recurrence and metastasis.
In this study, a rapid detection method based on time-resolved fluorescence microsphere immunochromatographic assay of Tg microspheres was established; we also verified its performance and conducted preliminary clinical comparisons.
  1. NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine,Jiangsu Institute of Nuclear Medicine. Wuxi, Jiangsu, China 214063.
  2. Department of Nuclear Medicine, DongFeng Affiliated Hospital of Hubei University of Medicine. Shiyan, Hubei, China
  3. Department of Nuclear Medicine, Shanghai 10th People’s Hospital, Tongji University School of Medicine, Shanghai, China
  4. . Department of Radiopharmaceuticals, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x
Materials and Methods
Data
A pair of antibodies (From Sigma-Aldrich Company, Building C, qiantan world trade center (phase ii), no. 3, lane 227, Dongyu road, Pudong new area, Shanghai, China) was used to capture the Tg present in samples. Goat anti-rabbit immunoglobulin and rabbit IgG (From Sigma-Aldrich Company, Building C, qiantan world trade center (phase ii), no. 3, lane 227, Dongyu road, Pudong new area, Shanghai, China) were used as quality control to ensure the accuracy of test results. Thyroglobulin antigen (From Roche Group, 1100 Longdong Road, Pudong new area, Shanghai, China) was used to substitute tissue in the experiments. Fluorescent microspheres (From Thermo Fisher Scientific, 27 Xinjinqiao road, Pudong new area, Shanghai, China) bind to antibodies and were used for detecting antigen–antibody binding. Analyzing membrane, water absorbing paper, sample pad, and binding pad (From GE,1 Yongchang North Road, Beijing, China) were included in the testing system. The test samples were obtained from the pathological examination of tissue types confirmed by the pathology department. Thyroid and lymph node tissues with metastatic thyroid cancer were included in the positive group, and muscle, fiber, fat, and other tissues were included in the negative control group.
Methods
After ultrasonic resuspension, 1 mL of the fluorescent microspheres were placed in a 1.5 ml EP tube and centrifuged at room temperature for 15 min at a centrifugation speed of 1×104 r/min. Further, 0.02 mol/L 2-(N-morph) ethanesulfonic acid(MES) buffer was used. The pH was washed from 2.0-8.0 twice and the 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) activation solution containing 2 mg/mL was added. After being activated at room temperature in darkness for 30 min, washing with 0.02 mol/L phosphate buffer (PBS), ultra-pure water, 0.05 mol/L HEPES buffer or 0.05 mol/L Tris-HCl buffer was performed twice. The above liquid was added according to the microsphere: the mass of the antibody was 1-20 μg, and the labelled antibody was added and conjugated for 2 h in darkness. After the binding was completed, a sealant equivalent to 1/20 of the volume of the coupling solution was added. The sealant was 0.02 mol/L PBS buffer containing 10% bovine serum albumin (BSA), ultra-pure water, 0.05 mol/L HEPES buffer or 0.05 mol/L Tris-HCl buffer. The buffer system of the sealant was the same as that of the binding buffer system. The reaction was allowed to proceed for 30 min in darkness. After centrifugation for 15 min, the supernatant was discarded and washed twice with deionized water. Then, the supernatant was redissolved with 0.02 mol/L PBS buffer containing 1% BSA (pH 7.4), and the above liquid was diluted to 1%-10% of the original concentration using 0.02 mol/LPBS buffer containing 1% BSA and 0.1% polysorbate and then stored as sample reaction solution at 4℃.(Figure1. Labeling of fluorescent microspheres)
NC membranes were laid on the bottom plate of polyvinyl chloride (PVC), and the coated antibody and quality control antibody were diluted to 0.1-1 mg/mL using 0.02 mol/L PBS solution (pH 7.4) containing 1% sucrose. The two were evenly sprayed on the NC film at an interval of 0.5 cm and 1.25 L/cm to form a detection line (T) and quality control line (C). The diluent was sprayed on the binding pad with a quantity of 150 μ L per 1.5 × 30 cm2. After 4 h of drying at 30℃ in the drying box, we laid absorbent paper and sample pads on both sides of the NC film. At 35% humidity, the test strip was cut into 0.4 cm-wide strips at 25℃ and put into the clamp to form the test strip.( Figure2. The composition of speed measuring card
)
The above reagent allowed to warm to room temperature. Tg antigen (approximately 100 ng/mL) was considered to be a high-value sample, and normal saline was considered to be a zero sample. Further, 50 μL of Tg antigen was added to the reagent strip and 50μL of sample reaction solution was added. The ratio of high-value sample/zero sample was defined as signal-to-noise ratio, and the method with the highest signal-to-noise ratio was selected as the optimal scheme.(Figure3. Testing Flow)
Statistical analyses
SPSS 19.0 software was used to analyze various performance indices of Tg-POCT kits and to assess the specificity and sensitivity of clinical tissue samples.
Results
During the research and development process, we found that pH and the amount of antibody had a great impact on the quality of the reagent, so we assessed the effect of the main indicators of each key link in the production process.
Effect of pH of activated liquid
The pH gradient of the microsphere during activation was 2.0 –8.0, and the optimum pH was 4.5.(Figure4. effect of pH on activation)
Effect of the pH of the coupling liquid
The pH gradient of the microsphere in the conjugated antibody was 5.0–12.0, and the optimum pH was 8.0.(Figure5. effect of pH on coupling)
Effect of buffer solution
During microsphere coupling, the buffer systems were PBS buffer, ultrapure water, HEPES buffer, and tris-HCl buffer. PBS was the optimum buffer solution.(Figure6.effect of buffer solution)
Effect of amount of antibody for labelling
The amount of antibody used for labelling was 1—20 µg. The optimum amount of antibody was 10 µg.( Figure7. effect of antibody labelled amount)
Effect of amount of antibody for coating
The amount of antibody used for coating was 1—20 µg; 0.8 µg was the optimum amount of antibody for coating.( Figure8.effect of antibody coated amount)
Effect of dilution ratio of fluorescent microspheres
The dilution ratio gradient of fluorescent microspheres was 1%–10%, and the optimum dilution ratio was 3%.( Figure9.effect of dilution ratio of fluorescent microsphere)
Performance verification