Fine-Needle Pricking Test of the Parathyroid Gland during Thyroid Surgery in Predicting Parathyroid Function

Background Permanent hypoparathyroidism is a serious complication following total thyroidectomy plus central neck dissection (CND). How to evaluate the vascularization of the parathyroid gland in real time is a major concern of thyroid surgeons. This study aimed to evaluate the fine-needle pricking (FNP) test in predicting parathyroid gland function. Methods The FNP test was performed in patients undergoing total thyroidectomy plus CND between January 1, 2014, and December 31, 2019, to visualize the vascularization of the parathyroid glands. Patients were classified according to the number of parathyroid glands preserved in situ with excellent vascularity (PGPIEV) demonstrated by FNP: group 0 (without PGPIEV), group 1 (with one PGPIEV), group 2 (with two PGPIEV), group 3 (with three PGPIEV), and group 4 (with four PGPIEV). Results A total of 608 patients with four parathyroid glands underwent FNP testing during thyroidectomy. At least one PGPIEV was demonstrated by FNP testing in 581 patients who had intact parathyroid hormone (iPTH) levels in the normal range after the operation. The prevalence of hypocalcemia decreased from 77.8% in group 0 to 9.8% in group 4 (P < 0.001), and the incidence of hypoparathyroidism decreased from 44.4% in group 0 to 0% in groups 1–4 (P < 0.001). iPTH concentrations on postoperative day 1 were positively correlated with PGPIEV groups (increased from 14.58 ng/l in group 0 to 45.22 ng/l in group 4, P < 0.001). Conclusions The FNP test is a safe and reliable method to predict parathyroid function. One PGPIEV demonstrated by the FNP test rules out the possibility of patients developing hypoparathyroidism.

e main causes of hypoparathyroidism after total thyroidectomy are intraoperative damage to the parathyroid glands by trauma, inadvertent parathyroid gland removal, or devascularization. e extent of damage to the parathyroid glands is difficult to predict during surgery. Some studies proposed that half of one normal parathyroid gland can produce sufficient parathyroid hormone (PTH) [9,10]. In order to avoid permanent hypoparathyroidism, parathyroid autotransplantation can be performed to salvage the devascularized parathyroid gland [11][12][13][14][15][16]. Typically, the strategic decision on how to select one parathyroid gland for autotransplantation relies on the viability of the gland. "Dead" parathyroid (without blood supply) has much greater odds of resurrection after autotransplantation than that remaining in situ [17]. e fine-needle pricking (FNP) test is a simple tool for evaluating the blood supply of the parathyroid gland. However, no studies have yet assessed the reliability and safety of the FNP test.
us, the present study aimed to evaluate the use of the FNP test in predicting parathyroid gland function, the method to identify whether one parathyroid gland should be autotransplanted or remain in situ, and the absence of postoperative hypoparathyroidism in patients in whom the test could demonstrate good vascularization of at least one parathyroid gland.

Patients.
A retrospective review of patients with thyroid cancer who underwent total thyroidectomy plus ipsilateral or bilateral central neck dissection (CND) between January 1, 2014, and December 31, 2019, was conducted. e exclusion criteria were previous thyroidectomy, preoperative hypo-or hyperparathyroidism, and osteoporosis requiring calcium or vitamin D treatment.
e study protocol was approved by the Ethics Committee of the Affiliated Sir Run Run Shaw Hospital, Zhejiang University School of Medicine.

Surgical Procedure and FNP Test Protocol.
yroid surgery was performed by two surgeons or two surgical fellows under their direct supervision. All patients underwent direct laryngoscopy preoperatively for the evaluation of vocal cord mobility. An intraoperative neuromonitoring instrument (NIM-Response 3.0 System; Medtronic Xomed, Jacksonville, FL, USA) was used for all patients. After the operation, laryngoscopy was performed only in patients with postoperative hoarseness. e extent of CND was assessed according to the American yroid Association guidelines [18]. Bilateral CND involved the removal of the laryngeal, pretracheal, and both the right and left paratracheal nodal basins. Unilateral CND involved the removal of the prelaryngeal, pretracheal, and one paratracheal nodal basin.
yroid lobectomy was performed according to the "meticulous capsular dissection" concept [19]. e parathyroid glands were not routinely and deliberately exposed during thyroidectomy. In case a parathyroid gland was encountered during thyroid lobectomy, best efforts were made to preserve the gland in situ with its feeding vessel. CND was performed according to the "layer of thymusblood vessel-inferior parathyroid gland" concept, as described previously [20]. However, best efforts were made during the CND to search for and preserve the inferior parathyroid gland.
After thyroid resection and CND, all identified parathyroid glands were scored for viability from grade 0 (no vascularity) to 2 (excellent vascularity) using the FNP test. e preserved parathyroid gland was handled with forceps; then, the parenchyma was pricked with a 25G injection needle. e bleeding from the small hole in the gland was assessed. A bleeding score for the FNP was established as follows: FNP 0, the parathyroid has no blood oozing after the pricking, indicating the gland is not vascularized; FNP 2, the parathyroid has blood effusing out after pricking, and the blood oozing persists when the blood covering the gland is cleared by suction, indicating that the gland is well vascularized; FNP 1, the parathyroid is oozing blood but slowly and in small volume, which persisted even after wiping with a gauze, suggesting that the gland is partially vascularized (Figure 1).
Parathyroid glands with an FNP score of 1 or 2 were retained in situ. If the FNP score was 0, the gland was routinely autotransplanted into the sternocleidomastoid muscle.
e parathyroid gland preserved in situ with excellent vascularity (PGPIEV) score was calculated according to the number of parathyroid glands preserved in situ with FNP score 2. Subsequently, the patients were classified according to the PGPIEV number as group 0 (zero PGPIEV), group 1 (one PGPIEV), group 2 (two PGPIEV), group 3 (three PGPIEV), and group 4 (four PGPIEV). e standard postoperative protocol of the institution was followed for all patients. Patients were not routinely administered calcium after total thyroidectomy plus CND unless the patient developed symptomatic hypocalcemia. Calcium supplements containing 750 mg of calcium carbonate plus 60 units of vitamin D3 were administered orally twice daily to treat symptomatic hypocalcemia. A calcium gluconate injection was prescribed for persistent symptomatic hypocalcemia after oral calcium treatment.

Laboratory Assays.
Serum calcium and intact parathyroid hormone (iPTH) levels were assessed in patients before the surgery and every morning (06.00 a.m.) thereafter until discharge. Most patients were discharged on postoperative day (POD) 3, depending on the volume of drainage fluid. Serum iPTH levels were measured on a Roche Cobas E601 instrument (Hitachi High-Technologies, Tokyo, Japan) (normal range 15-65 ng/l). e serum calcium levels were measured on an Abbott Aeroset Automated Instrument Analyzer (Toshiba Medical Systems, Tochigi-ken, Japan) (normal range 2.11-2.52 mmol/l). Ionized calcium amounts were not determined separately in this study.

Definition of Hypoparathyroidism.
Transient hypoparathyroidism was defined as an iPTH level below the normal range (15 ng/l) after the surgery [21][22][23]. Hypocalcemia was defined as the serum calcium level <2 mmol/l (8 mg/dl) or the requirement for calcium supplementation to treat the clinical symptoms of hypocalcemia, such as distal digital paresthesia or tetany, during the hospital stay [10,24]. Permanent hypoparathyroidism was defined as subnormal serum iPTH levels, calcium levels <2 mmol/l, or a requirement for calcium and/or vitamin D supplements to treat hypocalcemia-related symptoms for >6 months.

Statistical Analysis.
Continuous data were presented as mean ± standard deviation. e normal distribution of quantitative variables was assessed using the Kolmogorov-Smirnov test. e comparison of proportions was investigated using the Pearson's χ 2 test. A one-way analysis of variance (ANOVA) or the Kruskal-Wallis test was used for quantitative variables. P < 0.050 indicated a statistically significant difference. e data were analyzed using SPSS version 16.0 (IBM, NY, USA).

Patient Characteristics.
A total of 807 consecutive patients underwent total thyroidectomy plus CND in the clinical treatment group of Dr. Xie at the Department of Head and Neck Surgery between January 1, 2014, and December 31, 2019. A flowchart of the study is illustrated in Figure 2. Of these, 608 patients whose all four parathyroid glands were identified during the operation were included in the analysis. Table 1 provides the clinical data of the 608 patients who underwent total thyroidectomy plus CND, followed by the parathyroid gland FNP test.

FNP Test Results for Parathyroid Glands.
e results of the FNP test for parathyroid glands are presented in Table 2. Of the 608 patients who underwent FNP testing, 581 had an FNP score of 2 for at least one parathyroid gland. e PGPIEV group 0 included 27 patients, of whom 12 developed transient hypoparathyroidisms (Figure 3  . a-5, the blood covering the gland was cleared by suction. At a-6, blood was still oozing out from the incision of the gland. (b) A moderately well-vascularized parathyroid gland (FNP of score 1). b-5, the blood covering the gland was wiped by gauze. b-6, small and slow blood oozing from the incision of the gland was detected (black arrow). (c) A devascularized parathyroid gland (FNP score of 0). c-5, no blood oozing out from the incision of the gland waited for 10 to 20 seconds after the pricking. c-6, no blood oozing out after more times pricking of the gland.
Total tyroidectomy plus CND n = 807 Insufficient data n = 11 Less than four parathyroid glands identified n = 143 Included in the analysis n = 608 Serum level of iPTH above or less than the normal range before operation n = 45      Fine-needle pricking (FNP) score: FNP 0, the parathyroid has no blood oozing after the pricking, indicating the gland is not vascularized; FNP 2, the parathyroid has blood effusing out after the pricking, indicating that the gland is well vascularized; FNP 1, the parathyroid has blood oozing, but it is slow and small, and the slow blood oozing persist when it was wiped by gauze, suggesting that the gland is partially vascularized.

4) (Figures 3(c)-3(e), Figures
S1c-S1e supplementary information). erefore, in the 581 patients with at least one parathyroid gland with a FNP score of 2, postoperative iPTH levels were in the normal range. e serum iPTH concentration at POD 1 and its proportion of the initial value (before operation) are the indicators of the function of the in situ preserved parathyroid glands. e patients in PGPIEV group 0 had the lowest POD 1 iPTH levels (14.58 ng/l), while patients in PGPIEV group 4 had the highest POD 1 iPTH levels (45.22 ng/l) (Figure 3, Table S1 supplementary information). e iPTH levels on POD 1 were positively correlated with the PGPIEV score (P < 0.001) ( Table 3). e proportion of iPTH levels on POD 1 of the initial value (before operation) increased significantly from 42.7% in the PGPIEV group 0 to 114.9% in group 4 (P < 0.001) ( Table 3). erefore, the PGPIEV score was positively correlated with the function of in situ preserved parathyroid glands. 21/27 (77.8%) patients developed hypocalcemia in the PGPIEV group 0. Asymptomatic hypocalcemia was detected in 15 (60.0%) patients in PGPIEV group 1, in 15 (26.8%) patients in PGPIEV group 2, in 18 (9.8%) patients in PGPIEV group 3, and in 31 (9.8%) patients in PGPIEV group 4 (Table 3). Transient hypoparathyroidism was detected in 12 (44.4%) patients in PGPIEV group 0. None of the patients in PGPIEV groups 1-4 developed hypoparathyroidism (Table 3). A low PGPIEV score was correlated with a high prevalence of transient hypocalcemia (P < 0.001) and hypoparathyroidism (P < 0.001) ( Table 3).

Adverse Reactions and Follow-Up.
e FNP test did not give rise to any adverse reactions. e active bleeding by incision of the parathyroid gland parenchyma after the FNP test was stopped without needing hemostasis to control the bleeding. After > 6 months of followup, none of the 608 patients developed permanent hypoparathyroidism.

Discussion
e present study has shown that the FNP test of the parathyroid gland in patients undergoing total thyroidectomy plus CND is safe, and the results suggest an excellent correlation between FNP score and parathyroid function. e postoperative iPTH levels were normal in all patients who had at least one well-vascularized parathyroid gland, according to the FNP test. us, the FNP test is a reliable tool to decide whether one parathyroid gland should be autotransplanted or left in situ.
Currently, postoperative hypoparathyroidism is the most common complication following total thyroidectomy plus CND [25]. Especially, permanent hypoparathyroidism increases the risk of malignancy [7] and death [8], leading to cerebral, vascular, ocular, and renal damage and a significant decline in the quality of life [26,27]. Parathyroid identification and preservation in situ with an optimal vascular supply is the mainstay of safe thyroid surgery. However, some studies reported that parathyroid autotransplantation is an effective method to reduce the risk of permanent postoperative hypoparathyroidism, but is associated with a markedly high prevalence of transient hypoparathyroidism [11][12][13][14][15][16]28]. erefore, a reliable tool that can help the surgeon decide about the autotransplantation of one parathyroid gland during thyroidectomy is essential.
Most thyroid surgeons rely on visual inspection of a parathyroid gland to decide whether it is well-vascularized or whether it should be autotransplanted. However, observation of the parathyroid color change is not reliable [29]. High rates (4.1-16.2%) of permanent hypoparathyroidism have been reported after total thyroidectomy and CND in some thyroid centers [4][5][6]30].
e FNP test enables a direct evaluation of the blood supply to the parathyroid gland and assists in selecting patients who require parathyroid autotransplantation when a nonvascularized parathyroid gland is identified. In this study, parathyroid glands with an FNP score of 0 were routinely autotransplanted into the sternocleidomastoid muscle, and those with an FNP score of 1 or 2 were retained An early and accurate predictor of hypoparathyroidism is critical as it improves the selection of patients for early discharge and decreases the rate of readmission. e estimation of the iPTH level at different time points after thyroidectomy is the most commonly used method to evaluate parathyroid function [23,31]. However, the positive predictive value of the absence of hypoparathyroidism reported by other authors was no prospects [32,33]. Moreover, considering the cost, the assay to estimate the serum iPTH level was not available anytime in most hospitals; also, it was time-consuming. A rapid intraoperative parathyroid hormone assay at different time points may be used for clinical research but is difficult for routine clinical practice.
Parathyroid gland angiography with indocyanine green fluorescence has been reported as a reliable method to predict parathyroid function [21,22]. Nonetheless, it needs an expensive laparoscopic imaging camera system, which is not routinely available in most hospitals, and administering intravenous indocyanine green is a contraindication for iodine allergy patients. e FNP test is a safe, simple, and reliable method to predict parathyroid function during thyroid surgery in real time. In this study, we only included patients with all four parathyroid glands identified during thyroid surgery. Strikingly, 25 patients had only one wellvascularized parathyroid gland as demonstrated by the FNP test. e postoperative iPTH levels in these patients were in the normal range. Together, these findings suggest that having at least one FNP score of 2 for the parathyroid gland rules out the possibility of patients developing hypoparathyroidism and symptomatic hypocalcemia. e shortcoming of the FNP test is the identification of parathyroid glands with high accuracy. Even the most experienced thyroid surgeons can misinterpret other anatomical structures, such as thyroid and thymus nodules or lymph nodes, as a parathyroid gland. If good vascularization of nonparathyroid tissue was demonstrated by the FNP test, it would lead to a false reassurance that the patients would not develop hypoparathyroidism. e immunochromatographic test strip to detect fine-needle aspirated tissue PTH using the immune colloidal gold technique for rapid intraoperative parathyroid identification [34] maybe a promising method to assist surgeons in identifying parathyroid glands with certainty.
In conclusion, this study demonstrated that when at least one parathyroid gland was well-vascularized after total thyroidectomy plus CND, the postoperative iPTH levels were within the normal range in all patients. e PGPIEV score shows an excellent correlation with postoperative parathyroid function. e FNP test could be a safe, simple, and reliable tool for assessing the function of parathyroid glands, following thyroid resection in real time and deciding whether one parathyroid gland should be autotransplanted or retained in situ during thyroid surgery.
Data Availability e data that support the findings of this study are available in the following link: https://figshare.com/s/ 5e96bb2c405d3dd1723a.

Conflicts of Interest
e authors declare that they have no conflicts of interest.