The Potential Role of Platelet Indices and Red Cell Distribution Width in Metabolic Dysfunction-Associated Fatty Liver Disease

. Background: Hepatic steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma are all possible outcomes of metabolic dysfunction-associated fatty liver disease (MAFLD). Some platelet function measures are strongly correlated with the incidence of insulin resistance's intensity and its associated problems . Platelet indices, including platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), red cell distribution width (RDW), and red cell distribution width to platelet ratio (RPR), were found to be associated with the presence of many diseases. Hence, this research aimed to evaluate the significance of platelet indices and RDW in MAFLD and their possible association with the degree of liver steatosis and fibrosis.


Introduction
A group of international specialists has advocated replacing the pessimistic "nonalcoholic fatty liver disease" (NAFLD) with the more upbeat "metabolic dysfunctionassociated fatty liver disease" (MAFLD).Those with metabolic syndrome and an abnormal buildup of fat in the liver, regardless of alcohol consumption, are diagnosed with MAFLD [1].Hepatic steatosis alone is insufficient for diagnosing NAFLD [2].

Original research
Obesity, type 2 diabetes, or a metabolic imbalance are also required for a definitive diagnosis of NAFLD.NAFLD and MAFLD are on the rise in Egypt, and the country's high obesity rates are likely to be blamed.NAFLD and MAFLD were found to occur in 57.65% of the Egyptian sample population [3].Hepatic steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma are all disorders that fall under the umbrella of NAFLD [4].
For this purpose, a liver biopsy has been the highest standard for identifying liver pathology for many years; however, owing to its intrusive nature, expensive cost, and risk of severe consequences (bleeding in particular), non-invasive diagnostics were developed.Different scores are produced.The results of these tests can come from a wide range of noninvasive imaging modalities and combinations of serologic markers [5].Transient elastography (TE) utilizing ultrasound and a variable attenuation parameter has developed as a promising imaging technology in the last decade.
However, owing to the unavailability of the Controlled attenuation parameter (CAP) and the high cost of the device, there is an urgent need for a simple, cheap, available, and non-invasive precise test to detect the stages of liver fibrosis and steatosis [5].
Platelets are best known for their role in blood clotting; Platelets have dual physiologic roles as both cellular mediators of thrombosis and immune modulatory cells [6].The inflammatory state created in MAFLD associated with diminished thrombopoiesis results in an increase in MPV.This altered platelet is called the proinflammatory phenotype that enhances sinusoidal endothelial leucocyte recruitment, leading to the propagation of the inflammatory process in MAFLD.They play an essential part in the liver's inflammatory response.Leukocytes are recruited through the hepatic sinusoids after activating effector cells There was an association between increased RDW and platelet indices and fibrosis progression and the severity of inflammation.So MPV, PDW, RDW, and RPR may be used as non-invasive indicators for liver fibrosis and steatosis in MAFLD.
Evidence shows they had a high sensitivity and specificity for diagnosing NASH [19].
So, this study aimed to evaluate the significance of platelet indices and RDW in MAFLD and its possible association with the severity of liver steatosis and fibrosis.

Patients and Methods:
Two hundred twenty patients with MAFLD were recruited from the Tropical Medicine department at Tanta University for this cross-sectional study.From September 2021 until August 2022.Just after obtaining ethical approval from the Ethical Committee of the Faculty of Medicine, Tanta University (approval code: 34908/ 9 /21).This research included 220 patients,86 males and 134 females, more than 18 years old who were diagnosed with MAFLD when hepatic steatosis was present in addition to the presence of three or more of the following risk determinants: 1) increased waist circumference (>102 cm [>40 in] for men, >88 cm [>35 in] for women); 2) elevated triglycerides (≥150 mg/dl); 3) low HDL cholesterol (<40 mg/dl in men, <50 mg/dl in women); 4) hypertension (≥130/≥85 mmHg) or taking the antihypertensive drug; and 5) impaired fasting glucose (≥110 mg/dl) or taking antidiabetic drug [20].
We excluded patients who had one or more of the following conditions: age < 18 years, alcohol consumption (more than 40g of alcohol per day ), a history of viral hepatitis, chronic liver disease due to drug administration, autoimmune hepatitis, history of Original research blood diseases (e.g., ITP), history of myocardial infarction, and stroke, history of receiving drugs which cause steatosis ( e.g., amiodarone ) or drugs that interact with normal platelet functions (aspirin), anemia either microcytic hypochromic (iron deficiency anemia) or macrocytic due to vit B12 deficiency, history of blood transfusion during the last four months, or patients who refused to participate in this study.
All the patients were subjected to a complete history and examination.The examination which included body mass index and waist circumference, and laboratory investigation, which included liver functions, blood urea, serum creatinine, lipid profiles, and iron profile to exclude iron deficiency anemia) complete blood picture, which included platelet indices and RDW.
Venous blood samples were collected into sterile standard tubes with a consistent anticoagulant dose.After obtaining blood samples, laboratory tests were conducted a few minutes later in the Clinical Pathology department.The automated analyzer was used to perform the complete blood count analyses.
Ultrasound on the abdomen and pelvis and Transient Elastography (Fibroscan) were done for all the patients for evaluation of the liver condition.

Results
The study population consisted of 220 subjects, which included 86 ( Regarding MPV value, there was a significant increase in MPV as the steatosis grade increased.It was 10.87±0.92 in S1, 10.98±1.72 in S2, and 11.77±0.62 in S3, with p <0.001.PDW% also showed a significant increase as the steatosis grade increased as it was 61.09±7.12 in S1, 61.25±13.07inS2, and 68.45±3.69 in S3, with p <0.001.Also, we found that RDW% showed a significant increase when the steatosis grade increased, as it was 15.91%±1.51 in S1, 17.71±3.87inS2, and 19.95±2.86 in S3 with p<0.001.Also, RPR was higher when the steatosis grade increased, while PCT% showed no significant difference in its level about the steatosis grades; the p-value was 0.266, as shown in Table (3).
As regards the stages of fibrosis, there was a significant decrease in platelet count as the fibrosis stages increased (p<0.001) as well as MPV, PDW%, RDW%, and RPR significantly increased as the fibrosis stage increased (p<0.001).At the same time, PCT showed no significant difference in fibrosis stages, as shown in Table (4).

Original research
There was a significant positive relationship between MPV, PDW%, RDW%, RPR LDL, TG, and waist circumference (P<0.001).Still, there was a significant inverse relationship between them and HDL (p<0.001), as shown in Table (5).
There was a significant inverse relationship between platelet count and LDL, triglycerides, and waist circumference (p<0.001),but there was a significant positive relationship between it and HDL.There was no significant relationship between PCT and HDL, LDL, TG, and waist circumference, as shown in Tab (5).

Discussion
MAFLD is a new term that changed from NAFLD.MAFLD is a common chronic liver disease globally, associated with the growing obesity epidemic today  The current study found that NAFLD with advanced fibrosis had a higher red cell distribution width-to-platelet ratio (RPR).This agrees with the Zhou et al. study; their results showed a significant relationship between RPR and advanced fibrosis in the NAFLD population [39], which indicated that the RPR ratio might be used as an independent risk factor for advanced fibrosis.
In our study, there was a significant positive relationship between MPV and LDL, triglycerides, and waist circumference (P<0.001).Still, MPV and HDL had a significant inverse relationship (p<0.001).This agrees with Li L, who found that MPV was positively correlated with TC, TG, and LDL-C while negatively correlated with HDL-C for NAFLD patients [40].
Conclusions: Certain aspects of a patient's complete blood count could be used to help in the diagnosis of MAFLD and NAFLD.MPV, PC, RDW, and RPR were all significant indicators for the grades of steatosis, suggesting that RDW, platelets, and platelet indices could be used as non-invasive markers for diagnosing MAFLD.

[ 7 ]
. Several research studies suggest that changes in platelet function and shape may occur in diabetic patients and metabolic syndrome patients [8].The mean platelet volume (MPV), the platelet distribution width (PDW), and the plateletcrite (PCT) are used to measure platelet health and activity.Platelet indices have been hypothesized to be closely connected to the prevalence and severity of insulin resistance [9,10].When assessing erythrocyte size variation (i.e., anisocytosis), the red cell distribution width (RDW) is a valuable metric to use [11].RDW has recently gained importance Original research as a prognostic marker for a wide range of diseases and conditions, including sepsis, acute myocardial infarction, heart failure, autoimmune diseases, liver diseases, and various cancers [12-17].Also, RPR has gained substantial attention as a prognostic marker of various medical conditions such as severe burn injury, primary biliary cholangitis, patent ductus arteriosus, predicting hepatic fibrosis and cirrhosis in chronic hepatitis B, diagnosis of premature ovarian insufficiency; myocardial infarction; acute pancreatitis in pregnancy [18] because it is simple, easy to measure and handle, cost-effective, and accurate for predicting the severity of fibrosis.
Transient elastography was used to assess the staging of liver fibrosis and steatosis by measuring the velocity of a low-frequency (50 Hz) elastic shear wave propagating through the liver.This velocity has a direct relation to the stiffness of the tissue.The CAP score (Controlled attenuation parameter) is measured in decibels per meter (dB/m) and ranges from 100-400 as follows: S0:<237.7,S1: 237.7-259.4,S3(severe steatosis) :>292.3.Fibrosis was classified according to the following cutoff levels (F): F0 = no fibrosis (5.5 kPa), F1 = mild fibrosis (5.5 to 8.0 kPa), F2 = moderate fibrosis (8.0 to 10.0 kPa), F3 = severe fibrosis (11 to 16.0 kPa), and F4 = cirrhosis (>16.0 kPa) [21].Statistical analysis of the data Data were fed to the computer and analyzed using IBM SPSS software package version 20.0.(Armonk, NY: IBM Corp).Categorical data were represented as numbers and percentages.For continuous data, they were tested for normality by the Kolmogorov-Smirnov.Quantitative data were expressed as a range (minimum and Original research maximum), mean, standard deviation, and median, usually distributed quantitative variables.At the same time, the ANOVA test was used to compare the different studied groups, followed by the post hoc test (Tukey) for pairwise comparison.The significance of the obtained results was judged at the 5% level.

[ 22 ,
23].The clinic pathological spectrum of MAFLD covers a mild-to-severe range from simple steatosis (SS) to NASH and then to NASH-related fibrosis or cirrhosis .The capacity to assess the extent of steatosis and fibrosis of the liver and to estimate the course of the disease is essential in managing patients with MAFLD.For many years, the gold standard for this purpose was liver biopsy.However, its invasive nature, high cost, and risk of developing severe complications.New reliable blood markers required in the evaluation of liver fibrosis and clinical prognosis of cirrhosis are permanently in the interest of scientists.Several years ago, primary hematological indices used in everyday life were proposed as potential candidates in this area, e.g., MPV, PCT, and RDW, and the creation of diagnostic tools.These tools are composed of many scores obtained from multiple combinations of serologic markers and noninvasive imaging techniques [24].When a promising imaging modality emerged, ultrasound-based TE with CAP minimized sampling errors noticed with liver biopsy [ 25].This research aimed to evaluate the significance of platelet indices and RDW in MAFLD and their possible association with the severity of steatosis and fibrosis of the liver.Original research Gülali et al. imply that platelets contribute to the process of liver fibrosis by reducing the expression of the main fibrogenic cytokine TGF-β and upregulating the expression of matrix metalloproteinase [26].

Relationship between steatosis grades, platelet indices, and RDW % (n = 220).
SD: Standard deviation, RDW: red cell distribution width, PDW: platelet distribution width, PCT: plateletcrit, MPV: mean platelet volume, RPR: red cell distribution width platelet ratio grps: groups.p: p-value for comparing between [37]mi et al. revealed that lower PC (platelet count) is associated with more advanced fibrosis and found an inverse relationship between the progression of liver fibrosis and platelets; taking this into account, PC is presented in many prognostic scores for fibrosis and cirrhosis of the liver, this agrees our study there was a significant decrease in PC as the fibrosis stages and steatosis grade increased[27].Some other reports showed that patients with inflammatory bowel disease had increased RDW compared to health controls.Li et al.reported that anti-TNF antibodies, potent suppressors of inflammation, caused Original research complete resolution in hepatic steatosis in a mice model.This report shows that hepatosteatosis may be an inflammatory process[36].etal.also found an increase in RDW in patients with hepatosteatosis compared to healthy controls; this agrees with our study, as we found a significant positive correlation between RDW and fibrosis stages and steatosis grade[37].Also, Michalak A observed that elevation in RDW can be assumed as a sign of the progression of simple steatosis to steatohepatitis and the progression of liver fibrosis during MAFLD receptors, which increases [28].PDW directly refers to platelet size, changes with platelet activation, and reflects the heterogeneity in platelet morphology [29].Our study showed a significant positive correlation between PDW and fibrosis score and steatosis grade, which agrees with Milovanovic et al., who found that PDW is higher in NAFLD patients than controls [30].Still, the study of Cao et al. demonstrated a negative correlation between PC and PDW and the stage of fibrosis [31].Ozhan al. found that lower PC and higher MPV are independent indicators of MAFLD [32].Several studies have demonstrated that liver steatosis has been linked to the elevation in MPV [33].A major Korean study found a significant relationship between NAFLD and the increase of MPV values in 628 obese volunteers [34].This agrees with our research that showed a significant positive correlation between MPV and fibrosis stages and steatosis grade.