Thyroid Artery Flow Velocity and Serum Adenosine Deaminase After Methimazole Therapy in Autoimmune Hyperthyroidism

Summary

This protocol aims to assess thyroid vascularity and immune activity in autoimmune hyperthyroidism by integrating Doppler ultrasonography with serum adenosine deaminase (ADA) measurement. It provides a non-invasive approach to evaluate disease severity, monitor therapeutic response, and differentiate Graves’ disease from Hashimoto’s hyperthyroidism with improved clinical accuracy.

Abstract

Autoimmune hyperthyroidism, including Graves’ disease and Hashimoto’s hyperthyroidism, presents with overlapping biochemical features but distinct pathophysiological mechanisms. Non-invasive methods that reflect both vascular and immune activity may improve disease differentiation and treatment monitoring. This retrospective single-center study included 60 newly diagnosed hyperthyroid patients (30 Graves’ disease and 30 Hashimoto’s hyperthyroidism). All participants underwent baseline and 12-week follow-up evaluations after methimazole therapy. Assessments included thyroid function tests, superior thyroid artery Doppler parameters (peak systolic velocity [PSV], resistance index [RI]), and serum adenosine deaminase (ADA) levels. Statistical analyses included paired and independent t-tests and Pearson correlation. At baseline, PSV and ADA levels were higher in Graves’ disease compared to Hashimoto’s hyperthyroidism. After 12 weeks of methimazole treatment, both groups showed significant reductions in PSV and ADA (p < 0.05), with greater changes observed in Graves’ disease. Moderate correlations were observed between ADA levels and Doppler parameters. The combined assessment of Doppler ultrasonography and serum ADA provides a reproducible, non-invasive approach to evaluate vascular and immune changes during therapy. While the method shows potential for differentiating autoimmune hyperthyroid subtypes and monitoring treatment response, further validation in larger cohorts is required.

Introduction

Graves’ disease and Hashimoto’s thyroiditis are the most common reasons for hyperthyroidism globally. Their common immune systems do not explain the wide variety of symptoms and ways they develop in the body^1,2,3. This disease occurs when thyroid-stimulating hormone receptor antibodies cause the thyroid to grow and secrete excessive thyroid hormones. Unlike other thyroid disorders, Hashimoto’s thyroiditis causes the body’s defense cells, cytotoxic T cells, to destroy thyrocytes, contributing to transient hyperthyroidism because of released thyroid hormones in the inflammatory process.

In patients with hyperthyroidism, MMI is given first to stop TPO function and lower the body’s thyroid hormone production². It is well known that TSH treatment is effective, but current studies confirm that including other markers and imaging approaches can more confidently predict what issues the treatment may solve. Doppler ultrasonography of the thyroid arteries and ADA are gaining importance as safe ways to evaluate the condition and future of the disease^4,5.

This imaging method, which is both real-time and non-harmful to cells, supplies us with factual data on the blood vessels in the thyroid gland. In patients with Graves’ disease, PSV and RI from the STA and ITA have been strongly connected to thyroid function and blood vessel status^6,7. Higher PSV and lower RI are common symptoms caused by thyroid blood flow and hyperactivity of the gland. Hashimoto’s hyperthyroidism generally involves mild hemodynamic changes because it is an inflammatory condition and not one caused by excessive thyroid tissue⁸. They seem valuable for distinguishing hyperthyroid causes as well as for observing how treatment affects blood pressure and heart function.

Serum adenosine deaminase, which helps break down purines, is found in many lymphoid tissues and is used as an indirect sign of cell-mediated immunity^9,10,11. Autoimmune inflammation disorders (AITDs) and several other autoimmune and inflammatory diseases have shown an increase in ADA. Levels of ADA are high in Graves’ disease partly because of T-lymphocyte activity and cytokine release. Meanwhile, ADA levels in Hashimoto’s thyroiditis may be due to the build-up of certain immune cells and the destruction of follicles. Consequently, measuring ADA in the blood supports assessing immune activity, disease progression, and treatment effects¹².

Although these findings suggest progress, more studies are necessary to see if thyroid artery blood flow and ADA rise or fall during and after taking methimazole for both Graves’ and Hashimoto’s hyperthyroidism¹³. The existing literature is usually concerned with one disease or does not monitor in detail how these parameters develop after starting treatment. Moreover, by studying both the immune response and blood circulation after therapy with methimazole, we can see if real remission is happening or if the disease is still evolving.

This study has two important results for clinical care. It helps us understand how different disease types react to the same drug by examining thyroid artery flow and ADA levels over time in both Graves’ disease and Hashimoto’s hyperthyroid patients^14,15,16. Because of this, medical professionals can use biomarkers together with traditional thyroid function tests to guide treatment and outcomes in patients whose antibody or radioactive iodine results are uncertain or missing¹⁷.

For this reason, our study examines the differences in thyroid artery Doppler values (PSV and RI) and ADA levels during treatment and after 12 weeks of using methimazole in patients with Graves’ disease and Hashimoto’s hyperthyroidism. With a better understanding of their changes during treatment, we hope to achieve greater accuracy in identifying, predicting, and treating autoimmune hyperthyroidism, as well as finding useful parameter changes that can help guide individualized treatment plans.

This protocol aims to provide a reproducible method for integrating Doppler ultrasonography and serum ADA measurement to assess vascular and immune activity in autoimmune hyperthyroidism. The approach is particularly suitable in settings where radionuclide imaging is unavailable or contraindicated, and where repeated non-invasive monitoring is required. However, this method may be less suitable in patients with significant thyroid nodularity, prior thyroid surgery, or coexisting inflammatory or infectious conditions that may independently alter ADA levels.

Compared with standalone biochemical testing, the combined Doppler–ADA approach provides complementary functional and immunological information. Previous studies have shown that Doppler-derived PSV reflects thyroid vascularity, while ADA reflects T-cell–mediated immune activity^{18,19,20,21,22,23,24}. However, standardized protocols combining both parameters remain limited, which this study aims to address.

Autoimmune thyroid diseases (AITDs) are the primary cause of hyperthyroidism in most regions, including Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). Despite both being autoimmune in origin, GD and HT differ in their pathogenesis, clinical presentation, and treatment approaches. Because certain biomarkers are useful for diagnosis, disease monitoring, and treatment evaluation, increasing attention has been given to thyroid Doppler ultrasonography and serum adenosine deaminase (ADA) levels.

Graves’ disease occurs when thyroid-stimulating receptor antibodies (TRAb) activate the TSH receptor, leading to thyroid enlargement and excessive thyroid hormone secretion. Hashimoto’s thyroiditis most commonly results in hypothyroidism; however, in its early phase it can cause transient hyperthyroidism (Hashitoxicosis) due to the release of pre-formed hormones from damaged follicles^4,5,6. While TSH, FT₃, and FT₄ are essential for clinical assessment, they do not always distinguish between the causes of hyperthyroidism nor accurately reflect underlying disease activity⁷.

Doppler ultrasonography is now widely used to assess thyroid vascularity. Vitti et al. (1995) demonstrated that patients with Graves’ disease exhibit higher peak systolic velocity (PSV) and lower resistance index (RI) in the superior thyroid artery compared with individuals having Hashimoto’s thyroiditis or other thyroid disorders¹⁸. In GD, this pattern indicates increased blood flow and vascularity of the thyroid gland. Because thyroid blood flow reflects disease activity, Doppler parameters are especially valuable during the initiation of antithyroid therapy^18,19,20. Reference values for thyroid Doppler indices have now been established through multiple studies^21,22,23, allowing reliable comparison across different thyroid conditions. A post-treatment decline in PSV and normalization of RI have been correlated with biochemical euthyroidism and clinical improvement in Graves’ disease.

The flowchart given in Figure 1 illustrates sequential steps including patient screening, diagnostic classification into Graves’ disease and Hashimoto’s hyperthyroidism, baseline clinical and biochemical evaluation, Doppler ultrasonography of the superior thyroid artery, serum ADA measurement, methimazole therapy initiation, and 12-week follow-up assessment with repeat Doppler and biochemical analysis. This standardized workflow enables reproducible evaluation of vascular and immune responses during treatment.

Figure 1. Study workflow for evaluating thyroid vascularity and immune activity in autoimmune hyperthyroidism. The schematic diagram illustrates the sequential study design, including patient screening and eligibility assessment, diagnostic classification into Graves’ disease and Hashimoto’s hyperthyroidism, baseline thyroid function and autoantibody evaluation, superior thyroid artery Doppler ultrasonography, serum adenosine deaminase (ADA) measurement, initiation of methimazole therapy, scheduled follow-up visits, and repeat biochemical and Doppler assessment after 12 weeks of treatment. The workflow highlights the integrated evaluation of vascular and immunological responses during antithyroid therapy. Please click here to view a larger version of this figure.

At the same time, immune biomarkers such as serum ADA are of significant interest. ADA is essential for T-lymphocyte development and appears in elevated concentrations in various autoimmune and inflammatory diseases, including rheumatoid arthritis, systemic lupus erythematosus, and tuberculosis. Increased ADA levels in AITDs typically indicate heightened T-cell activity and reflect the severity of the autoimmune response against the thyroid²⁴.

Protocol

Ethics Statement

This study was approved by the Institutional Human Research Ethics Committee of Shishi City Hospital (Approval No.: 2026038). Written informed consent was obtained from all participants. All procedures were conducted in accordance with the Declaration of Helsinki.

1. Participant Recruitment and Enrollment

1. Screen Potential Participants
   1. Verify age between 18 and 60 years.
   2. Confirm newly diagnosed hyperthyroidism by suppressed TSH (<0.01 mLU·L^-1) and elevated FT3 and FT4.
2. Classify Participants into Diagnostic Groups
   1. Diagnose Graves’ disease by assessing for diffuse goiter, TRAb positivity, and/or characteristic diffuse hypervascularity on thyroid Doppler ultrasonography.
   2. Diagnose Hashimoto’s hyperthyroidism by confirming elevated TPOAb or TgAb, a hypoechoic glandular pattern on ultrasound, and absence of TRAb.
3. Apply Exclusion Criteria
   1. Exclude participants with prior antithyroid drug or steroid use.
   2. Exclude pregnant or lactating individuals.
   3. Exclude those with a history of thyroidectomy, radioiodine therapy, or nodular goiter.
4. Exclude Participants with Confounding Conditions
   1. Remove participants diagnosed with autoimmune or infectious diseases that may influence ADA levels.
   2. Exclude participants with inadequate ultrasonographic windows for Doppler assessment.
5. Enroll Eligible Individuals
   1. Enroll 30 participants with Graves’ disease and 30 with Hashimoto’s hyperthyroidism.
   2. Assign each participant a unique study identification code.

2. Baseline Clinical and Biochemical Evaluation

1. Collect Clinical Parameters
   1. Measure pulse rate, blood pressure, body mass index (BMI), and grade goiter size.
   2. Record symptom score based on predefined criteria.
2. Collect Fasting Blood Samples
   1. Instruct participants to fast overnight for at least 8 h.
   2. Draw 5–8 mL of venous blood under sterile conditions.
   3. Centrifuge blood samples at 1500 × g for 10 min at 4 °C to separate serum.
   4. Carefully transfer the supernatant serum into labeled microcentrifuge tubes without disturbing the cell layer.
3. Perform Thyroid Function Tests
   1. Measure TSH, FT3, and FT4 using standardized chemiluminescent immunoassays.
   2. Record results in the electronic database.
4. Assess Autoantibodies
   1. Measure thyroid receptor antibody (TRAb) levels to confirm Graves’ disease.
   2. Measure anti-TPO and anti-Tg antibodies for Hashimoto’s classification.
5. Measure Serum ADA Activity
   1. Use a kinetic UV method to quantify ammonia production.
   2. Express ADA activity in U·L^-1 according to assay instructions.
   3. Perform ADA assay using a kinetic enzymatic method based on ammonia production measured spectrophotometrically at 340 nm.
   4. Use an automated biochemistry analyzer with temperature maintained at 37 °C.
   5. Incubate reaction mixture for 5 min and record absorbance change per minute.
   6. Calculate ADA activity using calibration standards and express as U·L^-1.
      NOTE: Maintain serum samples at −80 °C if analysis is delayed.

3. Methimazole Administration and Follow-Up

1. Initiate Treatment
   1. Administer methimazole at 10–30 mg·day^-1 based on disease severity.
   2. Titrate dosage every two weeks according to biochemical and clinical response.
   3. Prescribe methimazole based on disease severity (FT4 levels and clinical symptoms).
   4. Allow beta-blocker use (e.g., propranolol ≤ 40 mg·day^-1) for symptomatic control; record usage.
2. Monitor Treatment Compliance
   1. Evaluate adherence using pill counts at each visit.
   2. Document symptoms and adverse drug reactions, including rash and hepatic dysfunction.
3. Schedule Follow-Up Visits
   1. Follow patients every two weeks for 12 weeks.
   2. Assess clinical improvement and biochemical normalization at each visit.
4. Repeat Biochemical Tests
   1. After 12 weeks of therapy, repeat TSH, FT3, FT4, and ADA measurements.
   2. Freeze serum aliquots at −80 °C if batch analysis is required.

4. Thyroid Doppler Ultrasonography

1. Prepare Patient for Examination
   1. Position the patient supine with neck extended and chin elevated.
   2. Apply an appropriate amount of ultrasound gel to the anterior neck.
2. Acquire Thyroid Artery Images
   1. Set pulse repetition frequency to 3–5 kHz and adjust the wall filter to a low setting. Maintain the Doppler angle at less than 60°. Position the sample volume at the center of the superior thyroid artery. Record peak systolic velocity over three consecutive cardiac cycles and calculate the mean value.
   2. Use a linear transducer (7–12 MHz) operated by a trained radiologist.
   3. Visualize the superior thyroid artery (STA) bilaterally in longitudinal view.
   4. Use a high-resolution ultrasound system with the following settings:
      1. Frequency: 7–12 MHz
      2. Pulse repetition frequency: 3–5 kHz
      3. Wall filter: 50–100 Hz
      4. Gain: Adjust to avoid background noise
      5. Sample volume: 1–2 mm
3. Perform Spectral Doppler Measurements
   NOTE: Confirm successful signal acquisition by obtaining a clear spectral waveform with well defined systolic peaks.
   1. Set the insonation angle to < 60°.
   2. Record peak systolic velocity (PSV) and resistance index (RI) using spectral mode.
4. Improve Measurement Accuracy
   1. Acquire Doppler readings for at least three consecutive cardiac cycles.
   2. Calculate the mean PSV and RI values for analysis.
5. Evaluate Hypervascularity
   1. Interpret STA as hyper-vascular if PSV ≥ 50 cm·s^-1 and RI ≤ 0.6.
   2. Record vascularity status in the study database.
6. Repeat Doppler After Therapy
   1. Perform Doppler ultrasonography six weeks after achieving euthyroidism.
   2. Compare pre- and post-treatment PSV and RI values.

5. Data Entry and Statistical Analysis

1. Prepare Data for Analysis
   1. Enter clinical, biochemical, and Doppler values into statistical software.
   2. Verify accuracy by performing double entry for 10% of samples.
   3. Perform statistical analysis using SPSS version 25.0 or equivalent statistical software.
   4. Select “Analyze > Compare Means > Paired-Samples T Test” for within-group analysis.
   5. Select “Analyze > Correlate > Bivariate” for Pearson correlation analysis.
2. Conduct Statistical Tests
   1. Use paired t-tests to compare baseline and post-treatment ADA and Doppler values within each group.
   2. Apply independent t-tests to compare values between Graves' and Hashimoto’s groups.
3. Assess Correlations
   1. Use Pearson correlation coefficients to determine associations between ADA, PSV, RI, and thyroid hormone levels.
   2. Consider p-values < 0.05 statistically significant.

6. Bias Control and Quality Assurance

1. Control Ultrasonographic Variability
   1. Assign all Doppler examinations to a single experienced sonographer.
   2. Maintain identical machine settings and protocols for all participants.
2. Ensure Laboratory Quality
   1. Perform daily internal quality checks for biochemical assays.
   2. Participate in external laboratory accreditation programs.
3. Maintain Blinding
   1. Blind the radiologist to participant diagnostic group.
   2. Blind laboratory personnel to clinical and Doppler findings.

7. Safety and Hazard Notes

1. Biological Sample Handling
   1. Wear gloves and protective eyewear during all blood collection and processing.
   2. Dispose of sharps in certified puncture-proof containers.
2. Chemical Hazards
   1. Handle ADA assay reagents containing phenol or ammonia-generating substrates using gloves and protective eyewear.
   2. Perform reagent preparation inside a chemical fume hood.
   3. Dispose of liquid waste in designated chemical waste containers according to institutional biosafety guidelines.
      CAUTION: toxic preservatives inside a chemical fume hood.
3. Pause Points: You may pause after serum separation by freezing aliquots at −80 °C. You may pause Doppler analysis by storing raw ultrasound images in DICOM format.
4. Dispose of biological waste (serum, blood) in biohazard bags and autoclave before discarding.
   1. Dispose of biological samples and reagents according to institutional biomedical waste management protocols. Treat all serum samples as potentially infectious and discard them in designated biohazard containers.
5. Dispose of sharps in puncture-resistant containers.

Results

A total of 78 patients were screened for eligibility, of whom 60 met the inclusion criteria and were included in the study, with 30 patients in each group. Eighteen patients were excluded due to incomplete clinical data or presence of confounding conditions. Doppler ultrasound measurements demonstrated high intra-observer reproducibility, with an intraclass correlation coefficient greater than 0.85. Serum ADA assays showed consistent results with minimal inter-assay variability (< 5%). Representative Doppler spectral ...

Discussion

The accuracy of this protocol relies primarily on two steps: standardized Doppler ultrasonography acquisition and precise ADA quantification. In Doppler imaging, the most critical factors are correct probe placement, maintaining a consistent angle of insonation (≤ 60°), and sampling the same segment of the superior thyroid artery (STA) in all participants. Incorrect angle correction or sampling from different arterial branches can markedly alter PSV and RI measurements. Similarly, proper fasting state and rest...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ADA (Adenosine Deaminase) Kinetic UV Assay Kit	Tulip Diagnostics		Kinetic UV method to quantify ADA (U/L)
Anti-Thyroglobulin Antibody (TgAb) Kit	Roche Diagnostics		Hashimoto’s hyperthyroidism marker
Anti-TPO Antibody Assay Kit	Roche Diagnostics		Used to identify Hashimoto’s thyroiditis
Automated Chemistry Analyzer	Beckman Coulter		For ADA testing (if run on chemistry platform)
Chemiluminescent Immunoassay Analyzer	Roche Diagnostics		Used for TSH, FT3, FT4 measurements
Methimazole tablets	Abbott		Antithyroid medication, 10–30 mg/day dosing per protocol
SPSS Statistical Software	IBM		Used for statistical analysis (SPSS v25)
TRAb Assay Kit	Roche Diagnostics		For diagnosis of Graves’ disease
Ultrasound Machine with Color Doppler	GE Healthcare		Used for thyroid Doppler evaluation, (LOGIQ series)