An overlooked situation in the interpretation of serum thyroglobulin level in a papillary thyroid cancer patient


 In the present study we report a case of thyroglobulin (TGB) measurement interference in a total thyroidectomized and radio-ablated 61-year old woman with papillary thyroid cancer. We investigated possible interference in the measurement of TGB due to discordant TGB in relation to clinical condition during the follow-up period. Serum TGB was measured with the chemiluminescence method using Beckman Coulter Unicel DxI 800 instrument. To investigate possible interference in TGB measurement serial dilutions, polyethylene glycol precipitation (PEG), treatment with heterophile blocking tube (HBT), rheumatoid factor level determination and retesting of TGB with an alternative method were performed. Serial dilutions of the serum sample revealed linearity but a remarkable decrease in TGB in the patient’s serum samples post PEG and post HBT treatments. Also, TGB results under functional sensitivity level obtained with a different method suggested that TGB interference developed due to heterophile antibody presence in the serum sample. The patient had unnecessarily undergone expensive imaging techniques, and invasive procedures such as lymph node fine needle aspiration biopsy, before the analytical interference was suspected by the clinician. This report illustrates the importance of early communication and close collaboration between clinicians and laboratorians in order to avoid unnecessary clinical intervention.

Abstract: In the present study we report a case of thyroglobulin (TGB) measurement interference in a total thyroidectomized and radio-ablated 61-year old woman with papillary thyroid cancer. We investigated possible interference in the measurement of TGB due to discordant TGB in relation to clinical condition during the follow-up period. Serum TGB was measured with the chemiluminescence method using Beckman Coulter Unicel DxI 800 instrument. To investigate possible interference in TGB measurement serial dilutions, polyethylene glycol precipitation (PEG), treatment with heterophile blocking tube (HBT), rheumatoid factor level determination and retesting of TGB with an alternative method were performed. Serial dilutions of the serum sample revealed linearity but a remarkable decrease in TGB in the patient's serum samples post PEG and post HBT treatments. Also, TGB results under functional sensitivity level obtained with a different method suggested that TGB interference developed due to heterophile antibody presence in the serum sample. The patient had unnecessarily undergone expensive imaging techniques, and invasive procedures such as lymph node fine needle aspiration biopsy, before the analytical interference was suspected by the clinician. This report illustrates the importance of early communication and close collaboration between clinicians and laboratorians in order to avoid unnecessary clinical intervention.
Nowadays, the immunometric second generation high-sensitive TGB (hs-TGB) assay was developed to accurately measure TGB at low concentrations with a functional sensitivity of 0.1 μg/L. These hs-TGB assays replaced the costly and uncomfortable TSH-stimulated TGB testing during follow-up of low-risk patients. TGB levels below 0.1 μg/L in athyrotic patients on suppressive levothyroxine therapy indicate a minimal risk (<1-2%) of clinically detectable recurrent papillary/follicular thyroid cancer and a high chance of being free of disease [3][4][5].
Here, we report a case with high level of TGB that is incompatible with her clinical condition due to interference of heterophile antibodies. We discuss the present case with the findings in the literature up to now.

Patients and methods
The patient was a 61-year old woman, with diagnosis of papillary thyroid carcinoma.
She was total thyroidectomized and radio-ablated with 100 mci I 131 in 2010. Since that time, she was followed by suppressive treatment with levothyroxine (LT4). She was admitted to Endocrinology and Metabolism Outpatient Clinic for her follow-up visits, which consisted of clinical examination with neck US and serum TGB determination between February 2014 and August 2015. In that period, serum TGB levels of the patient were found to be <0.1 μg/L and neck US was normal, but the patient interrupted her follow-up visits for three years. In October 2018 when she was admitted to our hospital three years later, thyroglobulin antibody (anti-TGB) value was 1.4 kIU/mL (near to limit of detection; 0.9 kIU/mL) and TGB value was 40.17 μg/L. This relatively high TGB value was confirmed by three subsequent tests on the same platform (36.54, 44.92 and 32.32 μg/L) on different days. During this process, thyroid stimulating hormone levels were (TSH) all below 0.5 mIU/L and anti-TGB were all negative. On neck US, no residual thyroid tissue was visualised but bilateral cervical lymphadenopathy was present. Due to the TGB positivity and suspicious US findings, the patient underwent fine needle aspiration biopsy (FNAB) and TGB was measured in the needle washout (FNAB-TGB). Also other imaging modalities such as thyroid scintigraphy (technetium 99m pertechnetate), whole body scanning using radioactive iodine (I 131 ), neck and thorax computed tomography (CT), and imaging with positron emission tomography (PET) were performed in order to eliminate local recurrence in cervical chain lymph nodes or distant metastases. All the investigations, other than the high serum TGB levels, ruled out recurrence and metastases of the disease. After all these tests were performed, communication between the endocrinologist and laboratory occurred on March 2019, and then interference studies were performed in the biochemistry laboratory.
For TGB interference studies, serial dilution, polyethylene glycol (PEG) precipitation, treatment with heterophile blocking tubes (HBT), RF measurement, and also analysis with a different manufacturer's instrument were performed.

Results
Firstly, serial dilutions (1/2, 1/4, 1/8) were performed on the suspicious serum sample using the manufacturer's diluent, which revealed linearity with a recovery of 95-105% and suggested no assay interference but we continued the investigation.
Secondly, PEG treatment was performed by mixing equals parts serum with a 25% (w/v) buffered phosphate solution of PEG 6000 (Merck, Darmstad, Germany). Samples were mixed and incubated at 4°C for 10 min and centrifuged at 14,000 g for 5 min, and then TGB measurement was performed with the supernatant obtained from this PEG-treated serum [6].
Thirdly, treatment of serum with HBT (Scantibodies Laboratory, Santee, CA, USA) was performed according to the manufacturer's instructions. Serum TGB levels were measured on the Beckman Coulter DXI 800 before and after treating serum samples with HBT.
Fourthly, due to the positivity for HAb, we performed an additional rheumatoid factor (RF) measurement using the immunoturbidimetric method, (Roche Cobas 6000, Roche Diagnostics, Mannheim, Germany). RF result was found to be 9.5 IU/mL and within the reference range (<14 IU/mL).
Lastly, the sample was sent to an external laboratory for determination of TGB on a different platform (Roche Cobas e601, Roche Diagnostics, Mannheim, Germany) using Elecsys TGBII reagent with electrochemiluminescence immunoassay method. Functional sensitivity for Elecsys TGBII of 0.040 μg/L. The TGB interference study results are summarised in Table 1.

Discussion
This report is about a rare case of a patient with a diagnosis of papillary thyroid carcinoma that showed false positive TGB levels during the follow-up associated with no clinical evidence of disease relapse.
Up to now, three possible causes of interference were identified in the measurement of TGB. The presence of TGB antibody may result in falsely low TGB measurement; therefore, it is recommended to analyse anti-TGB with TGB [1,3,4]. In our case, anti-TGB was negative in all the samples investigated. RF positivity was also shown to be a cause of false positivity in TGB measurement [7] but our patient had low RF levels (9.5 IU/mL) as well. The presence of HAb, a rare type of TGB interference [8], mostly causes a falsely high TGB result but also may cause a false negative result as well [8,9].
In our investigation, firstly we discovered that TGB measurements were performed with a Roche instrument during the time period of the early follow up (between February 2014 and August 2015) but in 2018 TGB measurements were performed with a Beckman Coulter instrument in our laboratory. Due to analysis with different assay and reagent antibodies, blood was sent to an external laboratory that used a Roche instrument. TGB negativity with the alternative method and the remarkable decrease in TGB in the patient's serum samples with post PEG and post HBT treatments suggest that HAb was the cause of discordant results. In our case only the serial dilution test did not support the presence of interfering antibodies, but it was reported that approximately 40% of samples containing interfering antibodies may fail to show a nonlinear relationship in the serial dilution test [10].
HAbs are human-developed antibodies to animal antigens. In immunometric methods, two antibodies that capture and mark the antigen are used. HAb reacts with both antibodies even though there is no antigen so they lead to a false result as if there is a high antigen level. This type of interference occurs in more than 90% of cases with HAb interference. More rarely, in less than 10% of HAb interference cases, they cause low antigens to appear [8]. False positive TGB measurements due to the presence of HAb may cause unnecessary investigations and RAI ablation treatments in differentiated thyroid cancer.
In our case, TGB interference developed due to HAb presence in serum samples and the patient had unnecessarily undergone expensive imaging techniques, and also invasive procedures such as lymph node FNAB, before the analytical interference was suspected by the clinician. In order to solve this problem, Giovenalla et al. suggested repeating the TGB testing using a different assay as the simplest way to reveal positive HAb interference, and also the use of HBT tubes for all sera referred for TGB measurement should be considered in order to prevent both unnecessary investigations or therapy, and delayed diagnosis of recurrence in patients with DTC [9].

Conclusion
Early communication between clinicians and laboratorians is enormously important before further investigations