Risk Factors for Granulocytopenia in Patients with Graves’ Disease Receiving Antithyroid Drugs

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Introduction
Te prevalence of hyperthyroidism is 0.2-1.3% in regions with sufcient iodine supply [1], and recent data have shown that the prevalence of hyperthyroidism in China is up to 0.78% [2]. Te causes of hyperthyroidism include Graves' disease (GD), toxic multinodular goiter, and toxic thyroid adenoma, among which GD is the most common. Te major treatments for GD include antithyroid drugs (ATD), 131I therapy, and surgery. ATD is the primary treatment of GD. Long-term ATD therapy is cost-efective, has favorable effcacy without damaging thyroid tissues, and causes few complications. It presents a certain advantage in improving patients' quality of life and other biological outcomes [3]. However, adverse drug reactions (ADR) may occur during administration, including pruritus, liver injury, granulocytopenia, and agranulocytosis. Granulocytopenia is defned as a neutrophil count (NEUT) <2.0 × 10^9/L. Agranulocytosis is defned as NEUT <0.5 × 10^9/L, commonly manifests as sore throat, high fever, and pneumonia; skin infections or sepsis may occur in severe cases [4]. Agranulocytosis usually occurs 2-3 months after administration and has a low incidence (about 0.1-0.5%), but once it develops, the mortality rate can be up to 4.0-6.3% [5]. Terefore, patients who developed granulocytopenia during the treatment of GD with ATD were included to further study the risk factors. Raising awareness of this group of patients in clinical practice, further preventing granulocyte defciency by predicting risk factors, and providing a reference for the early detection of high-risk patients, are the issue to be explored in this study.

Research Subjects.
We retrospectively examined that patients who were diagnosed with GD and treated with ATD (Department of Endocrinology, Nanjing Drum Tower Hospital, January 2010-July 2022) were recruited as subjects.
Te inclusion criteria were as follows: (1) patients diagnosed with Grave's disease according to the Chinese Guidelines for diagnosis and Management of Hyperthyroidism and Other Causes ofTyrotoxicosis [5]; (2) patients receiving regular antithyroid therapy with methimazole (MMI) and propylthiouracil (PTU) for more than a week; (3) no abnormality observed in patients' NEUT prior to ATD therapy.
Te exclusion criteria were as follows: (1) patients with a previous history of immune system and hematologic diseases; (2) granulocytopenia due to exposure to radiation toxins, chemoradiotherapy, or antibody therapy; (3) acute granulocytopenia (also known as transient granulocytopenia) caused by infection, infammation, etc.; (4) patients who did not receive drugs regularly or switched to surgery or 131I therapy halfway through the treatment; (5) patients with large amounts of missing data.
Grouping: Patients were assigned to observation and control groups based on the occurrence of granulocytopenia. Granulocytopenia (observation group) was defned as NEUT <2.0 * 10^9/L in routine blood tests during ATD treatment, and patients with granulocytopenia induced by malignancy, immune system diseases, hematologic diseases, or other drugs were excluded. Patients in the control group did not present with granulocytopenia during ATD administration.

Statistical Analysis.
Statistical analysis was performed using SPSS.26.0. Normally, distributed continuous data were expressed as mean values ± standard deviations (SD), and comparisons between groups were conducted using parametric tests. Nonnormally distributed continuous data were expressed as median values and interquartile range (IQR), and comparisons between groups were performed using nonparametric tests. Categorical data were expressed as n (%) and analyzed using the χ 2 test. Signifcant indicators in the univariate analysis were included in the multivariate logistic regression analysis to determine the factors for granulocytopenia in patients receiving ATD, with the adjusted OR values and 95% CI calculated. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was acquired. AUC >0.5 represented predictive value, and P < 0.05 stood for statistical signifcance.

Multivariate Analysis of Factors for Granulocytopenia
Induced by ATD. Te occurrence of granulocytopenia was regarded as the dependent variable, and the eight signifcant factors in the univariate analysis were included as independent variables. Binary logistic regression analysis was performed after assigning values to variables. Male was assigned a value of 0 and female was assigned a value of 1. According to the low value below the normal range, the normal range, and the high value above the normal range, the values of WBC, NLR, FT3, FT4, and TSH were divided into three categories. ALT and AST were divided into two categories with normal values as the dividing line, with values of 0 and 1, respectively. Te results showed that sex, NLR, ALT, and WBC count were independent risk factors for granulocytopenia; female sex and ALT elevation were risk factors; NLR and WBC elevation were protective factors ( Table 2).

Te Diagnostic Values of Gender, ALT, WBC, and NLR.
Females and higher ALT indicated positive results, that is, granulocytopenia, presenting low diagnostic values; lower NLR and WBC indicated positive results. NLR had an excellent predictive performance for granulocytopenia in patients receiving ATD (AUC � 0.916, sensitivity � 89.5, specifcity � 80.4). Te results of the ROC analysis are shown in Table 3.

Discussion
Granulocytopenia occurs in approximately 10% of newly diagnosed and untreated GD patients [5]. ATD does not need to be discontinued when NEUT >1.5 * 10^9/L and granulocyte count could recover after administering WBCelevating drugs for a certain period. However, inappropriate management can lead to further development of agranulocytosis, which greatly increases the risk of infection and can even be life-threatening. Terefore, it is crucial to analyze risk factors for ATD-induced granulocytopenia.

Risk Factors for Granulocytopenia in Patients Using ATD
4.1.1. Gender. Tis study revealed that sex might be a risk factor for ATD-induced granulocytopenia. A retrospective study in Japan showed that among all patients with Graves' disease [6], a greater number of women were treated with ATD to develop granulocytopenia (p < 0.05). Although hyperthyroidism is more common in females, adverse reactions caused by drugs are more related to individual factors, such as physiological conditions and genetic factors. Research has shown that females have almost double the risk of ADR compared with males, possibly because diferent populations have diferent pharmacokinetics [7]. Terefore, the efect of gender on ATD-induced granulocytopenia requires further investigation.

ALT.
Reactive metabolites are produced during the ATD oxidation process to activate infammasomes to induce immune responses, thereby destroying neutrophils [8]. Tis oxidation process is mediated by myeloperoxidase and cytochrome P450, which might explain why some drugs that induce granulocytopenia or agranulocytosis are hepatotoxic [4]. Te current study demonstrated that patients with elevated ALT levels were more likely to have agranulocytosis; however, further investigations are required to validate this fnding.

WBC.
Granulocytes are a type of WBC, and thyrotoxicosis can lead to reductions in the WBC count and granulocytes [9]. However, some studies have suggested that routine WBC monitoring may be the most efective predictor of agranulocytosis, secondary to ATD [10]. Studies from Japan also recommended WBC monitoring once every two weeks [11]. Multivariate regression analysis confrmed that reduction in WBC count was a risk factor for granulocytopenia. Tere is still no consensus about the necessity of regularly monitoring WBC and NEUT for early identifcation of ADR, but the guidelines recommend routine monitoring. Patients should be advised to discontinue ATDs if they develop fever, sore throat, or mouth ulcers during treatment and to have their blood tested immediately.

NLR.
Te NLR is an infammatory marker used as a prognostic indicator for the recurrence and survival of patients with cancer [12]. Meanwhile, research has shown that NLR can assist in the diferentiation of hyperthyroidism with diferent etiologies [13]. In this study, the NLR was included as a variable. Te ROC curves showed that NLR performed well in predicting granulocytopenia with higher sensitivity and specifcity than sex, ALT, WBC count, etc. Terefore, close attention should be paid to NLR when administering ATD to avoid aggravation of granulocytopenia. Taken together, sex, ALT, WBC, and NLR are signifcant predictors of ATD-induced granulocytopenia. Early identifcation of granulocytopenia is conducive to improving patient prognosis, reducing drug-induced ADR, and ofering a reference for devising timely protocols in clinical practice. Due to the retrospective nature of the study, some data were missing, resulting in a small sample size available for analysis; the low incidence of ATD-induced granulocytopenia led to the low number of patients in the observation group available for analysis. Hence, a prospective study with a large sample size is required to further probe the risk factors for ATD-induced granulocytopenia, and mechanisms should be delved into to provide theoretical support for early diagnosis and prevention.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Te study was performed in accordance with the Helsinki Declaration. Approval was granted by the Medical Ethics Committee of Nanjing Drum Tower Hospital of the Afliated Hospital of Nanjing University Medical School (No. 2022-285-03).

Consent
Informed consent was obtained from all individual participants included in the study.

Disclosure
Jiaxi Li and Xiaowen Zhang should be considered the joint frst author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.