上海第六人民医院陆汉魁主任带领的小组做了用免疫胶体金（ICG）来测量促甲状腺激素（TSH）水平的研究，这是一种快速，简单易行的手段，而且可以在实验室外进行。本研究旨在将中国首个以ICG为基础的定性定量TSH测量方法的临床适用性与世界范围内通用的TSH测量金标准——第三代血清TSH化验进行对比。
对283个初次就诊的疑似甲状腺功能减低症的对象采集指尖血和静脉血，且两种血在半小时内采集完成。指尖血用ICG定性定量法来测量TSH水平，静脉血则使用第三代血清TSH化验的方法。不同测量系统测得结果的相关性用一致率kappa值和Spearman相关系数来检测和表示。
以第三代血清TSH化验为金标准，ICG定性测试盒的kappa值为0.86，用来筛查甲减的敏感度为85.00%，特异度为99.38%。所有对象中，假阴性和假阳性的所占比例分别为6.38%和0.35%，而这两种方法的总一致率为93.26%。同样以第三代血清TSH化验为参照，ICG定量TSH分析体统的Spearman相关为0.91，敏感度88.43%，特异度98.77%。使用定量系统产生假阴性和假阳性的比例分别为4.95%和0.71%，总的一致率为94.35%。
两种基于ICG的测量系统均比以实验室测量为基础的第三代血清TSH化验简单和快速，且在对甲减的简便筛查中有着不错的表现。
Clinical Evaluation of the Immune Colloidal Gold Method for Rapid Qualitative and Quantitative Measurement of Thyroid-Stimulating Hormone as an Assay for Hypothyroidism
Tingting Wang . Shiwei Sheng . Meifang Ruan . Jing Yan .Jianying Gu . Yumin Jiang . Yunchao Gao . Hankui Lu
Introduction: The immune colloidal gold (ICG) method of measuring thyroid-stimulating hormone (TSH) is a rapid and easy-to-perform test, allowing off-site measurements. This study compared the clinical utility of the first ICG-based qualitative and quantitative TSH test methods in China with the third-generation serum TSH assay used worldwide.
Methods: Fingertip and venous blood was collected within 30 min from 283 patients initially suspected of hypothyroidism. TSH was measured in fingertip blood using ICG-based qualitative quantitative tests. Serum TSH in venous blood was tested using the third-generation serum TSH assay. Correlations between systems were tested by kappa or Spearman correlation coefficients.
Results: Compared with the third-generation serum TSH assay, the ICG-qualitative TSH test kit had a kappa coefficient of 0.86, a sensitivity of 85.00%, and a specificity of 99.38% in screening for hypothyroidism. The percentages of false negatives and false positives among all subjects were 6.38% and 0.35% respectively; the total consistency rate of the two methods was 93.26%. When compared with the third-generation serum TSH assay, the ICG-quantitative TSH analysis system had a Spearman correlation coefficient of 0.91, a sensitivity of 88.43%, and a specificity of 98.77%. The percentages of false negatives and false positives among all subjects were 4.95% and 0.71%, respectively; the total consistency rate of the two methods was 94.35%.
Conclusion: Both ICG-based assays are easier and faster to perform than the third-generation, laboratory-based serum TSH assay method. The ICG-based methods showed acceptable performance in the simplified screening for hypothyroidism.
Trial registration: ClinicalTrials.gov identifier,NCT01921452.
Funding: Merck Serono Co., Ltd.
Keywords: Hypothyroidism; Immune colloidal gold; Thyroid-stimulating hormone;Thyrotropin
The diagnostic strategy for evaluating thyroid function has focused on measuring serum levels of thyroid-stimulating hormone (TSH), as immunoassays for TSH are more sensitive and more reliable than immunoassays for free thyroxine (FT4) and/or triiodothyronine (FT3) in evaluating both hypothyroidism and hyperthyroidism [1–3]. TSH is the main regulator of thyroid hormone biosynthesis and secretion [4]. In many clinical circumstances,TSH is initially assayed to simplify the diagnosis of thyroid dysfunction in ambulatory patients.This is especially applicable in patients at high risk of clinical or subclinical hypothyroidism,including older patients, female patients, and those with a personal or family history of thyroid disorders [5].
Surveys of many regions have shown high rates of hypothyroidism and subclinical hypothyroidism [6–9]. In China, thyroid function tests are presently performed only in hospital laboratories at a high cost, with considerable time elapsing until patients receive test results because of heavy laboratory workloads [10]. In addition, primary care units cannot assess thyroid function as they do not have the appropriate laboratory equipment[11]. The availability of point-of-care testing of thyroid function screening would provide more rapid results for high-risk patients in primary and secondary care practices as well as geriatric units. Simple tests of TSH levels in fingertip blood, similar to tests for early pregnancy that measure human chorionic gonadotropin levels[12] and tests of glucose levels to screen for hyperglycemia in patients with diabetes mellitus [13], may play a role in the diagnostics and screening for suspected hypothyroidism.
One similar method available in China to measure TSH uses the immune colloidal gold (ICG) technique to label the anti-TSH antibody for qualitative and quantitative measurement of TSH in whole finger blood rather than serum[14]. This study aimed to evaluate the clinical utility of the ICG-based qualitative and quantitative TSH tests as compared with the third-generation serum TSH assay routinely used in clinical practice [15].
Study Subjects
This was a single-center, open-label, interventional trial. Inclusion criteria of study population were the following: patients with suspected hypothyroidism; willing to comply with the study protocol; signed an informed consent form. Subjects were excluded if they were menstruating, had bleeding hemorrhoids or hematuria, had taken aspirin within 48 h of enrollment, or were unable to understand or
cooperate with the study. All subjects were included in the full analysis set.
Instruments and Reagents
The ICG-qualitative TSH test kit and ICG-quantitative TSH analysis system were purchased from Tianjin Xinwan Biological Technology Co., Ltd. (Tianjin, China). The third-generation serum TSH assay, an automated electrochemiluminescence immunoassay (ECLIA) system, was obtained from Roche Diagnostics GmbH (Mannheim, Germany) and was regarded as the gold standard instrument for comparisons.
Principle of Colloidal Gold Rapid Diagnostic Tests
Testing Principle for the ICG-Qualitative TSH Test Kit
Figure 1 shows a schematic of the ICG-qualitative TSH test strip. Blood added to sample hole “S1”passes through the porous filtration system as a result of longitudinal capillary action. The blood cells are blocked by the top layer, while the liquid component, plasma, reaches the bottom layer of the membrane. Buffer added to buffer hole “S2”spreads along the bottom layer of the membrane and mixes with the plasma and other dispersed substances on the bottom layer. If the plasma TSH level is higher than the level of detection, TSH will bind to the colloidal gold-labeled dye conjugate to form TSH-dye complexes. These complexes are captured by the solid-phase chromogenic agent located in the test zone “T” on the membrane to form a pink ribbon. Other coloring substances will be captured by the quality control zone “C” to show a clear pink ribbon, a positive control for the TSH test. The kit can be used for testing and evaluating whole blood samples with TSH levels≥5.00μIU/ml.
Fig. 1 Colloidal gold rapid test strip. S1 is the sample hole; S2is the buffer hole
Testing Principle for the ICG-Quantitative TSH AnalysisSystem
The ICG-quantitative TSH analysis system consists of aPILOT-110R analyzer, a test board, a radiofrequency identification tag, data managementsoftware (version no. V1.1.0x, KAIWOOD Technology Co., Ltd., Taiwan, China),and buffer. The testing principles are identical to those of the TSH test kitdescribed above. Test results are analyzed automatically by the PILOT-110Ranalyzer, radiofrequency identification tag, and data management software, witheach assay calibrated internally.
Sampling
Two drops of fresh whole blood were drawn from eachsubject’s fingertip using a disposable blood sampling needle. The blood wastaken up by the 30-μl capillary pipetteand added to the sample hole on the reaction panel, and TSH buffer was added30–90 s later. Following incubation for 10 min at room temperature, thequalitative TSH results were determined by observation with the naked eye. Alternatively,for the ICG-quantitative TSH analysis system, the reaction panel was incubatedfor 15 min at room temperature and inserted into the computing channel. TSHlevels were calculated by the system and displayed on the screen.
To avoid the TSH fluctuations that may be causedby pituitary secretion cycles [16,17], fingertip and venous blood samples were collectedwithin 30 min of each other. After collecting fingertip blood samples,＞1.0 ml venousblood was drawn from each subject, and serum TSH was measured by the third-generationserum TSH assay using theECLIA method (ECLIA serum TSH assay). Generally, serumTSH levels could be determined within 4 h of blood drawing.
Diagnostic Criteria
As stated by the manufacturer, both the ICG-qualitativeTSH test kit and the ICG-quantitative TSH analysis system were calibrated inaccordance with Diagnostic Products Corp. Immunoassay System
(Immulite 2000). The cutoff value on the ICG-qualitativeTSH test was 5.00μIU/ml, with levels≥5.00μIU/ml indicative of potential subclinicalor clinical hypothyroidism. In the result types of the ICG-qualitative TSH testkit shown in Fig.2, a positive result is defined as theappearance of two pink ribbons, one each inzone “C”and zone “T”, suggesting thatthe TSH levels are≥5.00 μIU/ml. A negative resultis defined as the appearance of a pink ribbon in zone “C”but none in “T”, suggestingthat the TSH levels are＜5.00μIU/ml. An invalidresult is defined as the absence of pink ribbons in both zones or the appearanceof a pink ribbon only in zone“T”, invalidresults may be caused by reagent deterioration or improper performance of theassay. The ICG-quantitative TSH analysis system has a functional detectionrange of 0.50–20.00μIU/ml. Again, thecutoff value for possible subclinical or clinical hypothyroidism was≥5.00μIU/ml.
Fig. 2 The result types of the ICG-qualitative TSH test kit. S1 isthe sample hole; S2 is the buffer hole; T is the test zone; C is the qualitycontrol zone
In our routine clinical practice, subclinicalhypothyroidism was defined as serum TSH≥4.20μIU/ml, as determined by the ECLIA serum TSHassay, plus normal serum thyroxine, whereas clinical hypothyroidism was definedas serum TSH≥10.00μIU/ml with reduced serumthyroxine, with or without relevant clinical signs and symptoms. Diagnostic thresholdsfor TSH are listed in Table1.
Statistical Analysis
SAS 9.2 software (Cary, NC, USA) was used for allstatistical analyses. Endpoints and 95% confidential intervals (CIs) werecalculated. Consistency hypothesis tests were adopted for both TSH test results(negative or positive) and TSH levels. The relationship between the ICG-qualitativeTSH test results and ECLIA serum TSH assay was evaluated by determining thekappa coefficient, whereas the relationship between the ICG-quantitative TSHanalysis system results and ECLIA serum TSH assay was evaluated by determiningthe Spearman correlation coefficient. Performances of the ICG-based methods ascompared to the gold method were evaluated by the sensitivity, specificity, false-negative,false-positive, and consistency rates.
Compliance with Ethics Guidelines
This study was registered at ClinicalTrials.gov, underidentification no. NCT01921452. The study protocol was approved by the Committeeson Ethics and Research Protocols of Shanghai Jiaotong University AffiliatedSixth People’s Hospital.
All procedures followed were in accordance with the ethicalstandards of the responsible committee on human experimentation (institutionaland national) and with the Helsinki Declaration of 1964, as revised in 2013.All subjects provided voluntary written informed consent prior to performing trial-relatedactivities.
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