The stent thrombosis in Belgium (STIB) scoring system reliability in Indonesia patients and the modified STIB scoring (M-STIB)

Clopidogrel is recommended as an alternative to Acetyl Salicylic Acid (ASA), the first-line drug for secondary stroke prevention. Clopidogrel resistance in Indonesia is reportedly 15.8%. The Stent Thrombosis in Belgium (STIB) is a scoring system proposed to assess clopidogrel resistance which has not been tested for the reliability in Indonesian population. The objective of the study is to test the reliability of the STIB scoring and modified-STIB scoring system in Indonesian population. This study was conducted cross-sectionally in Dr. Cipto Mangunkusumo Hospital and Universitas Indonesia Hospital from January 2020 to December 2021. Laboratory examinations of human CYP450 concentration in blood plasma from 112 subjects were carried out using the ELISA method. The clopidogrel resistance test was carried out using the VerifyNow method. Out of 112 ischemic stroke patients in this study, 14.3% of them did not respond to clopidogrel. Cross-tabulation between the STIB score and clopidogrel resistance showed significant results (p < 0.05) on the Hb variable and a combination of two or three factors involving Hb levels. Both Hb and CYP450 levels can be independent factors predicting clopidogrel resistance. Once combined, the Hb and CYP450 cutoff levels of 458.9 pg/mL had a sensitivity of 100% with a specificity of 30.2%. STIB scoring system did not prove to be reliable in screening for Clopidogrel resistance in Indonesian population. The modified STIB scoring system proposed in this study showed a promising result. Further research with larger population should be conducted.


Introduction
The leading cause of disability and death worldwide is stroke [1].In Indonesia, the 2013 Basic Health Research (Riskesdas) reported the prevalence of stroke was 7% and increased to 10.9% in 2018 [2].
A recurrent stroke is an acute vascular event after the stabilization of the previous stroke [3].The rate of recurrent stroke ranges from 7 to 20% in the first year and increases after 5 years by 16-35% [4,5].The recurrent stroke rate in Indonesia was 19.9% [6].To avoid stroke recurrence, adequate secondary preventive management is required, where oral antiplatelet drugs are recommended to be administered within the first 24-48 h after the first stroke onset [7,8].
Acetyl Salicylic Acid (ASA) is recommended as the first-line drug to prevent recurrent stroke (secondary prevention) [9].Clopidogrel is recommended as an alternative in cases of intolerance or contraindication to ASA [10].Clopidogrel is a prodrug that will be converted into active metabolites by cytochrome P450 in the liver.The active metabolite of clopidogrel will inhibit platelet aggregation induced by Adenine diphosphate (ADP) [11].ADP or its receptors may be affected by several factors that result in a decreased response to clopidogrel, which may alter the blockage of ADP receptors through several processes known as clopidogrel resistance [12].Clopidogrel resistance in Chinese-Americans is reported to be relatively high (68-73%), which is thought to be because nearly 50% of Asians are missing an allele of genetic function that can reduce the response to clopidogrel [13].
VerifyNow is light transmission aggregometry which frequently utilized to test clopidogrel resistance.However, it is relatively expensive in Indonesia, costing roughly $150.Stent Thrombosis in Belgium (STIB) is one of several scoring systems developed to measure clopidogrel resistance and is anticipated not to require a VerifyNow assessment.The STIB scoring system identifies a history of diabetes mellitus, a body mass index (BMI) of > 28 kg/m 2 , and hemoglobin (Hb) level below 13.9 g/dL as the three characteristics that predict clopidogrel resistance [14].The presence of two or three risk factors correspond to 62.6% or 77.8% clopidogrel resistance, respectively.Clopidogrel monotherapy is not an option if the STIB score indicates resistance [14].Due to the high expense of VerifyNow testing for clopidogrel resistance, the researchers conducted a study to measure the reliability of STIB scoring in Indonesian patients and attempted to modify the STIB scoring system.

Methods
The Neurology Outpatient Clinic of Cipto Mangunkusumo Hospital (RSCM) and Universitas Indonesia Hospital (RSUI) hosted the cross-sectional study in 2020 and 2021.The researchers employed a sequential sampling approach to choose the participants.Patients who experienced either a primary or recurrent ischemic stroke and had been administered clopidogrel for at least 5 days qualified for inclusion in this study.Patients with a history of clopidogrel allergy, impaired renal function, blood coagulation disorders, and certain medications administration, including omeprazole, esomeprazole, and atorvastatin, were excluded from the study (Fig. 1).Statistical analyses were conducted using IBM SPSS Statistics (Version 26) and presented descriptively.Bivariate analysis was performed to determine the correlation between various variables and CPG resistance.Chi-square analysis was conducted and p value of < 0.05 was deemed significant.
Human CYP450 analysis was conducted in Pharmacology Laboratory in Universitas Indonesia by the standards of the Indonesian Ministry of Health.CYP450 concentration was measured using ELISA method, which has high sensitivity and specificity  to detect human CYP450 [15].P2Y12 reaction units (PRU) with a cutoff of > 208 was deemed inadequate response (resistant).

Results
Based on 112 subjects' epidemiological characteristics of the studied patients (Table 1), it was found that in this study, most of the subjects were men (58.9%), < 68 years (88.4%), and with BMI < 28 (76.8%).Adjusting to the participant characteristics of this study, the researchers made additional cutoffs for age of > 57 years and BMI of > 25.
The hemoglobin level, BMI, and history of diabetes were cross-tabulated on the clopidogrel resistance data to create the STIB score variable.The STIB was included in the cross-tabulation component as the three factors crossed, two, and three factors combined.Significant correlations between clopidogrel resistance and Hb, Hb+BMI, and Hb+DM were found (p < 0.05) (Table 3).
This study proposed a modification of the STIB score by looking at several prognostic factors.The results of cross-tabulation of various prognostic variables against clopidogrel resistance with a p value of < 0.25 can be seen in Table 4. Based on the result, it was also found that there was a significant correlation (p < 0.05) between clopidogrel resistance and hemoglobin levels and CYP450 concentrations with a cutoff of 458.9 pg/mL.
To determine the correlation between prognostic variables and clopidogrel resistance, multivariate analysis in the form of linear regression was subsequently conducted on the prognostic variables with p value of < 0.25 (Table 4).Based on multivariate analysis, the hemoglobin (p = 0.003) and CYP450 cutoff of 458.9 pg/mL (p = 0.004) both had a significant correlation.
Between these two variables, each of them had the potential to independently predicts clopidogrel resistance.Hb 13.9 g/dL and CYP450 458.9 pg/mL were thus combined to test how the two variables performed as an assessment component in the modified STIB score (Table 5).The combined sensitivity and specificity of the Hb and CYP450 cutoff levels of 458.9 pg/mL were 100% and 30.2%, respectively.

Discussion
This study assessed clopidogrel resistance using Veri-fyNow.The mean and median yielded 115.2 PRU and 105.5 PRU, respectively.These results indicated that the subjects of this study have an excellent response to clopidogrel (85.7%), meaning that the Indonesian population is sensitive to clopidogrel administration.Only a few subjects (14.3%) did not respond to clopidogrel.Similar results were found in a preliminary study by Hidayat and colleagues [15] which the number of resistances was only found in 9 people (15.8%) [15].These results differed from the two previous studies, the STIB study in Belgium and the GRAVITAS study in the United States (50.2% and 40.8%, respectively) perhaps due to the difference in baseline characteristics and genetic makeup [16].
Most subjects were male (58.9%) and under 68 years (88.4%), with the median and mean being 57.5 and 57.14 years, respectively.The age restriction for the STIB score was 68 years (p 0.05), yet Belgium, the nation that created the STIB score, has an 81.75-year life expectancy.The mean age of this study was 57 years, while the average life expectancy in Indonesia was 71.2 years [2].The study also found that the subjects were dominated by a BMI score of < 28 kg/m 2 (76.8%).
It was found that 33% subjects in this study were diabetics.It was per the results of a previous study by Lau et al. [17], who reported that 1 in 3 stroke patients (24.7-56.2%)did not have diabetes.It contradicted the Indonesian registry data, where 24% of ischemic stroke patients have DM [18].
The BMI, Hb levels, and DM components comprised the STIB score.In other words, DM symptoms, BMI readings above 28 kg/m 2 , and hemoglobin levels below 13.9 g/dL predicted an elevated risk of clopidogrel resistance.According to a recent study by Nie et al. [19], BMI scores did not significantly correlate with clopidogrel resistance (p = 0.921).The study by Legrand et al. [20]  and Price et al. [21], which revealed a significant correlation, was distinct from the study.In contrast to the Indonesian population, which had a lower BMI, they obtained the mean BMI of the non-resistant group at 27 kg/m 2 , the unresponsive group at 28 kg/m 2 , the median score of the non-resistant group at 29 kg/m 2 , and the unresponsive group at 31 kg/m 2 .There was a difference that reached 3-4 kg/m 2 with the Indonesian population which might affect the BMI variable in the Indonesian population to not be a factor that affects clopidogrel resistance.
According to this study, the Hb level was an independent factor that could raise the risk of clopidogrel resistance by 19 times if it was ≤ 13.9 g/dL (p = 0.003).Previous findings confirmed that the average Hb level in the clopidogrel resistance group was 13.6 g/dL (p = 0.001) [20].
Our findings on the correlation of DM comorbidity and HbA1c level with clopidogrel resistance were in line with Liu et al. [22] and Nie et al. [19], who also found no significant correlation between the two variables (p > 0.05).The studies by Legrand and colleagues [14] and Price et al. [20], which demonstrated a significant correlation (p = 0.05), had different results.It was assumed that variations in the mean scores caused variance in the result.In this study, the non-resistant and resistant groups had mean HbA1C values of 6.6% and 6.3%, respectively, compared to 6.0% and 6.3% in previous study [14,20].
The cross-tabulation of the STIB score variable with clopidogrel resistance in this study showed that only Hb levels had a significant correlation (p < 0.001).In this study, the highest sensitivity was obtained by Hb levels   The STIB score was adjusted in this study to become the modified-STIB score (mSTIB) by adding CYP450 level as one of the components.Except for CYP450, which was determined to be involved in drug metabolism, none of the evaluated risk factors demonstrated a significant correlation with clopidogrel resistance.According to the results of the genetic analysis, the median score of CYP450 was 407.63 pg/mL, and the mean score was 874.5 pg/mL in the group with a normal response to clopidogrel.The synthesis of modified STIB may have been affected by CYP450 levels in this study (p = 0.005).The modification of Hb+CYP450 displayed a very high sensitivity in the STIB test.When paired with the Hb value, this study discovered a cutoff CYP450 concentration of 458.9 pg/mL with a sensitivity value of 100% and a specificity value of 30.2%.Given that CYP450 testing was reasonably inexpensive, these findings suggested that it can be recommended as one of the prognostic tests for clopidogrel users.However, the number of the subject was not adequate enough to conclude CYP450 as one of the factors to predict CPG resistance.

Conclusion
The STIB score was not reliable to screen for CPG resistance in Indonesian population.The STIB score was not advised for Indonesian patients with poor resistance rate unless they had a history of recurrent stroke, were taking medications regularly, and had no hematological or cardiac abnormalities.This study attempted to suggest a modified STIB scoring by adding CYP450 level of ≥ 458.9 pg/mL and showed a promising result.Further research should be conducted with larger number of subjects.

Table 1
Epidemiological and clinical characteristics of the studied ischemic stroke patients BMI body mass index, DM diabetes mellitus, SD standard deviation, min minimum, max maximum

Table 2
Clopidogrel resistance results and laboratory examinationsHbA1c hemoglobin A1c, FBG fasting blood glucose, HDL high-density lipoprotein, LDL low-density lipoprotein, AST aspartate aminotransferase, ALT alanine aminotransferase, SD standard deviation, min minimum, max maximum

Table 3
Cross tabulation results between STIB score and resistance variables BMI body mass index, Hb hemoglobin, DM diabetes mellitus, CI confidence interval *Chi-square; † p < 0.05

Table 4
Cross tabulation results between prognostic variables and clopidogrel resistance with p value < 0.25 FBG fasting blood glucose, AST aspartate aminotransferase, CYP450 cytochrome P450, OR odd ratio, CI confidence interval * Chi-square; † p < 0.05

Table 5
Relationship between independent prognostic variables on clopidogrel resistance