Complete Blood Count Inflammatory Ratios in Attention Deficit Hyperactivity Disorder Children

doi:10.21203/rs.3.rs-4438361/v1

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Complete Blood Count Inflammatory Ratios in Attention Deficit Hyperactivity Disorder Children

https://doi.org/10.21203/rs.3.rs-4438361/v1

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Background

Attention deficit hyperactivity disorder (ADHD) is considered the most common neuropsychiatric disorder in children. Many researchers have suggested that changes in pro-inflammatory and anti-inflammatory cytokines could play a role in the pathogenesis of ADHD. We aimed in this study to evaluate the relationships between the CBC inflammatory markers (NLR, MLR, PLR, and MPV) and the severity of attention deficit hyperactivity disorder symptoms in children. Method: This study was a cross-sectional case-control study carried out on 80 ADHD children and 40 apparently healthy children as the control group. A complete blood count was obtained (white blood cell (WBC), neutrophil count, lymphocyte count, monocyte count, eosinophil and basophil counts, platelets, and mean platelet volume (MPV). Based on these counts, the neutrophil lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), and monocyte lymphocytic ratio were calculated. Results: CBC inflammatory ratios (NLR, MLR, and PLR) were considerably higher among children with ADHD than in the control group; moreover, a significant positive correlation was observed between the CBC inflammatory ratios (neutrophil/lymphocyte, monocyte/lymphocyte) and ADHD symptoms severity assessed by Conner's parent rating scale scores (total ADHD score, hyperactivity, impulsivity, and inattention scores). Conclusions: The results of our study proved that inflammation plays a significant role in the intricate pathophysiology of ADHD and suggest that CBC inflammatory ratios can be used as an indicator for ADHD severity. This is easily obtainable and reliable in evaluating the role of inflammation in ADHD etiology.

ADHD

mean platelet volume

neutrophil/lymphocytic ratio

monocyte/lymphocytic ratio

platelet lymphocytic ratio

CBC

Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorders, affecting nearly 5.2% of children and 2.6% of adolescents (1, 2). ADHD has a negative impact on the lives of affected children, resulting in poor educational and work attainment, risk-taking behaviours, and unsatisfactory relationships (3, 4, 5, 6). Furthermore, significant comorbidity between ADHD and other mental illnesses has been reported, particularly for mood disorders, anxiety disorders, drug and alcohol use disorders, and autistic spectrum disorders (7, 8, 9). Although the definite cause of ADHD is unknown, complex interactions between genetic and environmental risk factors are responsible and play a significant role, with a heritability rate of about 70–80% (10). Many researchers suggest that a variety of environmental exposures during the prenatal stage raise the risk of ADHD in offspring (11). These results imply that many factors influencing pregnancy may contribute to the development of ADHD in the fetus. Many studies supported the presence of maternal immune activation and the immune-inflammatory alterations in children with ADHD (12, 13). According to the most recent meta-analysis, young people with ADHD had unaltered peripheral blood levels of IL-1β, IL-6, and IL-10 and a considerably low level of TNF-α (13). Furthermore, cytokines are crucial to neurodevelopmental processes as they affect glial cell formation as well as neural and synaptic maturation (14).

Alterations in different peripheral blood ratios, such as neutrophil to lymphocyte ratio (NLR), monocyte to lymphocyte ratio (MLR), and platelet to lymphocyte ratio (PLR), have been demonstrated to act as biomarkers of immune-inflammatory processes and are associated with poor outcomes in various diseases, such as cancer, cerebrovascular, cardiovascular, or autoimmune diseases (15). These ratios have also been studied and investigated in recent years in individuals suffering from mental disorders. It was observed that patients with bipolar disorder, depression, or non-affective psychosis had higher levels of these inflammatory markers (16, 17).

Recently, the link between NLR, PLR, MLR, and ADHD was investigated, but the findings have been inconsistent. According to much research, people with ADHD have higher inflammatory ratios, which may indicate that inflammation plays a significant role in the etiology of this condition (18, 19). On the other hand, additional research has not discovered any significant variations in these parameters among individuals with ADHD, indicating the intricacy of the immune-inflammatory processes implicated (20).

The current study's aim was to evaluate the role of the CBC inflammatory ratios NLR, MLR, PLR, and MPV as an easy and inexpensive method that can be assessed in ADHD disorder that could be applicable for routine use.

The study was a cross-sectional case-control study carried out at the Pediatric Department and Clinical Pathological Department, Minia University Hospital, on 120 children, whose age range was 6–15 years, during the period from April 2021 to April 2023. The diseased children were diagnosed to have ADHD according to the DSM-5 criteria (21). The Conners’ Parent Rating Scale was used to assess the severity of the ADHD symptoms (22).

The children included in this study were divided into groups as follows: Group 1 included 80 ADHD children on regular follow-up visits to the Pediatric Neuro-Psychiatric Outpatient Clinic of Minia University Hospital. Their age range was 6 to 15 years.

They were further divided into the following subgroups: Group 1A included 40 recently diagnosed ADHD children before receiving any medical treatment for ADHD. Group 1B included 40 children who had previously been diagnosed with ADHD and were under treatment with non-stimulant ADHD medication for at least 6 months.

Group 2 included 40 apparently healthy children whose age and sex matched those in Group 1. Subjects with other psychiatric or neurological diseases, hypercholesterolemia, hematological diseases, other acute or chronic physical illnesses, drug administration during the last month, or exposure to smoking were not included in the study.

Written informed consent was obtained from the participants' parents or legal guardians following an extensive explanation of the study to the participants. The study received approval from the Ethics Committee, and the patients' medical records were also reviewed.

All blood samples were collected via venous blood sampling by sterile venipuncture under complete aseptic conditions in an EDTA-containing tube for CBC analysis. The complete blood count was determined by an automated cell counter (CelltacES, Nihon Kohden, Germany), and the NLR, PLR, MLR, and MPV were calculated. The absolute platelet count divided by the absolute lymphocyte count yielded the PLR, and the absolute monocyte count divided by the absolute lymphocyte count yielded the MLR.

Statistical Analysis:

The statistical analysis was conducted using IBM SPSS for Windows Version 23. The Kolmogorov-Smirnov test was utilized to evaluate the data distribution. Descriptive statistics were utilized to present the means ± SD, medians (25–75 percentiles), and frequencies, expressed as percentages. For comparing two groups of independent variables with non-normal distributions, the Mann-Whitney U-test was applied. Spearman's correlation analysis was employed for non-normally distributed independent variables, while Pearson correlation analysis was used for normally distributed ones. A significance threshold of P < 0.05 was applied, indicating statistical significance. At the ideal cut-off point for every inflammatory marker, the corresponding sensitivity, specificity, positive, and negative predictive values were calculated (23, 24).

The mean age of Group 1A was 8.15 ± 2.62 SD; they were 90% males and 10% females; the mean age of Group 1B was 8.5 ± 1.67 SD; they were 70% male and 30% female, whereas the control group mean age was 9.15 ± 1.59 SD; they were 65% male and 35% female, and there was no significant difference among all studied groups as regards demographic data. Table 1

Table 1

Demographic characteristics of the studied groups
	Group 1				Group 2 (Control) (n = 40)		P value
	Group 1A (ADHD Un-medicated) (n = 40)		Group 1B (ADHD Medicated) (n = 40)		Group 2 (Control) (n = 40)
	Mean ± SD	Range	Mean ± SD	Range	Mean ± SD	Range
Age (yrs.)	8.15 ± 2.62	6–15	8.50 ± 1.67	6–12	9.15 ± 1.59	6–12	0.290
Sex: Male N (%) Female N (%)	36(90%) 4 (10%)		28 (70%) 12 (30%)		26 (65%) 14 (35%		0.155
Weight(kg)	28.30 ± 7.63	19–49	27.6 ± 5.86	19–46	30.70 ± 5.18	23–41	0.273
Height(cm)	130. ± 11.50	105–153	127. ± 8.09	115–151	130.35 ± 5.33	122–139	0.399
BMI	16.30 ± 1.83	12.70–20.90	16.80 ± 1.61	14.3–20.10	17.0 ± 4.41	0.19–22.10	0.745
ADHD: attention-deficit hyperactivity disorder BMI: body mass index.

There was no significant difference regarding the duration of ADHD illness in the unmedicated ADHD group and the medicated ADHD group. The family history of ADHD was positive in 25% of unmedicated ADHD children, while in the medicated group, the family history was positive in only 5%. 50% of the medicated group were treated with atomoxetine, and the remaining medicated children were receiving risperidone. Table 2.

Table 2

Comparison of the clinical data between un-medicated and medicated ADHD group:
Data of disease	Group 1A (Un-medicated) (n = 40)		Group1 B (Medicated) (n = 40)		P value
Data of disease	Mean ± SD	Range	Mean ± SD	Range	P value
Duration of illness (months)	17.9 ± 10.0	8–36	26.4 ± 10	12–36	0.106
Duration of treatment (months)	-	-	11 ± 6.88	1–24	-
Type of treatment: rispexidone(%) Atomaxateine (%)	-		20 (50%) 20 (50%)		-
Family history +Ve (%) -Ve (%)	10(25%) 30(75%)		2 (5%) 38 (95%)		0.077

The ADHD group showed significantly higher ESR values compared with the control group (P values = 0.0001 and 0.0001, respectively). There was a significantly higher lymphocytic count in the control group than in the unmedicated and medicated groups (P values = 0.0001 and 0.003, respectively); moreover, there was a significantly higher lymphocytic count in the medicated group than in the unmedicated group (P value = 0.018). The neutrophil count and neutrophil/lymphocyte ratio were significantly higher in the unmedicated group and medicated group than in the control group (P values = 0.0001, 0.003, 0.0001, and 0.006, respectively). There were no significant differences between the medicated and unmedicated groups regarding the neutrophil count and neutrophil/lymphocyte ratio (P values = 0.166 and 0.121, respectively). Table 3.

Table 3

Comparison of Inflammatory markers among the studied groups:
Laboratory data	Group 1		Group 2 (Control) (n = 20)	P value
	Group1 A (Un-medicated) (n = 20)	Group 1B (Medicated) (n = 20)	Group 2 (Control) (n = 20)
	Mean ± SD	Mean ± SD	Mean ± SD
ESR 1h(mmHg) Mean ± SD	21.8 ± 10.2	18.1 ± 7.82	8.05 ± 2.28	< 0.0001*
ESR 1h(mmHg) Mean ± SD	21.8 ± 10.2	18.1 ± 7.82	8.05 ± 2.28	1A&1B 1A&2 1B&2	0.276 0.001* 0.001*
CRP (mg/l) Median IQR	1.5 1–3	2.5 1–10.25	2 1.25–4	0.121
MPV% Mean ± SD	9.28 ± 0.82	9.41 ± 1.57	8.92 ± 1.01	0.4
Lymphocytes (%) Mean ± SD	35.5 ± 5.28	42.6 ± 10.3	51.3 ± 7.45	< 0. 001*
Lymphocytes (%) Mean ± SD	35.5 ± 5.28	42.6 ± 10.3	51.3 ± 7.45	1A&1B 1A&2 1B&2	0.018* 0.0001* 0.003*
Neutrophils (%) Mean ± SD	53.8 ± 5.19	48.9 ± 10.8	39.7 ± 7.94	< 0.0001*
Neutrophils (%) Mean ± SD	53.8 ± 5.19	48.9 ± 10.8	39.7 ± 7.94	1A&1B 1A&2 1B&2	0.166 0.0001* 0.003*
Monocytes% Median IQR	6 5–7	5 4.25–6	4 3–5	0.457

The monocyte/lymphocyte ratio was significantly higher in the unmedicated and medicated ADHD groups than the control group (P values = 0.0001 and 0.001, respectively), and it was also significantly higher in the unmedicated group than the medicated group (P value = 0.031). Moreover platelet/lymphocytes ratios were significantly higher in un-medicated group in comparison to control group (P value = 0.004), while platelet/lymphocytes ratios have neither significant difference between the medicated ADHD group and control groups nor between un-medicated and medicated ADHD groups (P values = 0.317 and 0.138, respectively). Table 4.

Table 4

Comparison of the CBC Inflammatory ratios in all studied groups:
Laboratory data	Group 1		Group 2 (Control) (n = 40)	P value
	Group1 A (Un-medicated) (n = 40)	Group 1B (Medicated) (n = 40)
	Mean ± SD	Mean ± SD	Mean ± SD
Neutrophil/ lymphocyte ratio ● Median ● IQR	1.4 1.25–1.87	1.1 0.825 − 1.7	0.88 0.6–1	< 0.0001*
				1A&1B 1A&2 1B&2	0.121 0.0001* 0.006*
Monocyte/ lymphocyte ratio ● Median ● IQR	0.17 0.13–0.22	0.11 0.1–0.14	0.06 0.052–0.08	< 0.0001*
				1A&1B 1A&2 1B&2	0.031* 0.0001* 0.001*
Platelet/ lymphocyte ratio Mean ± SD	138 ± 61.5	109 ± 38.0	88 ± 36.4	0.005*
				1A&1B 1A&2 1B&2	0.138 0.004* 0.317

In this study, we found that impulsivity, inattention, and total ADHD Conner's scores were significantly higher in the unmedicated group than the medicated group (P values = 0.048, 0.004, and 0.002, respectively), while there was no significant difference regarding the hyperactivity score between the unmedicated and medicated groups (P value = 0.075). Table 5. We also found a significant positive correlation between the CBC inflammatory ratios (neutrophil/lymphocyte, monocyte/lymphocyte) and ADHD symptoms severity evaluated by Conner's parent rating scale scores (total ADHD score, hyperactivity, impulsivity, and inattention scores), while the platelet/lymphocyte ratio had no significant correlation with hyperactivity and impulsivity (P values = 0.296 and 0.584, respectively), but it showed a significant positive correlation with total ADHD Conner's score and inattention only. Table 6.

Table 5

Comparison of ADHD Conner's scores between ADHD (un-medicated and medicated) groups
Scores	Group1 A (Un-medicated) (n = 40)	Group1 B (Medicated) (n = 40)	P value
Scores	Mean ± SD	Mean ± SD	P value
Hyperactivity score	55.4 ± 3.81	52.5 ± 6.12	0.075
Impulsivity score	54.1 ± 5.07	50.8 ± 5.15	0.048*
Inattention score	54.2 ± 3.04	50.5 ± 4.58	0.004*
ADHD score	55.1 ± 3.29	50.9 ± 4.35	0.002*

Table 6: Correlation between Conner’s ADHD scores and CBC inflammatory ratios:

Correlation between ADHD Conner’s scores and CBC inflammatory ratios		r value	p value
Hyperactivity score	Neutrophil lymphocyte ratio	0.394**	0.012*
	Monocyte lymphocyte ratio	0.324**	0.044*
	Platelet lymphocyte ratio	0.169	0.296
Impulsivity score	Neutrophil lymphocyte ratio	0.412**	0.008*
	Monocyte lymphocyte ratio	0.433**	0.006*
	Platelet lymphocyte ratio	0.089	0.584
Inattention score	Neutrophil lymphocyte ratio	0.361**	0.022*
	Monocyte lymphocyte ratio	0.368**	0.021*
	Platelet lymphocyte ratio	0.331**	0.037*
ADHD score	Neutrophil lymphocyte ratio	0.371**	0.019*
	Monocyte lymphocyte ratio	0.357**	0.026*
	Platelet lymphocyte ratio	0.372*	0.018*

In this study, there was no significant difference between all studied groups regarding demographic data and other CBC parameters (Hb, WBCs, and platelets); similarly to these results, Akinci and Uzun found that there were no significant differences between the ADHD group and the control group regarding the HB, WBCs, and platelet count (25). On the other hand, Fahiem et al. found that HB and RBC counts were significantly lower in the children with ADHD than the control group, and their lower values were linked with learning disabilities in ADHD children (26).

This study showed that there were significantly higher ESR values (an acute phase reactant that increases inflammation) in the ADHD unmedicated and medicated groups than the control group. This result was supported by the study of Sahu et al., who reported that ESR was significantly higher in the ADHD group compared to the control group (27). Their study showed no significant difference in the MPV between the ADHD (unmedicated and medicated) and control groups.

This result was similar to that of Garipardic et al., who found no significant difference in MPV values between the ADHD group and the control group (28). Contrary to our result, Yorbik et al. found that MPV was higher in ADHD patients than the control group and suggest that it may be used as a marker of the inflammatory reaction in patients with ADHD (29). Our study showed that the neutrophil count was significantly higher in the ADHD group than the control group, while there was no significant difference between the medicated and unmedicated ADHD subgroups. Also, there was a significantly higher lymphocytic count in the control group than in the unmedicated and medicated groups.

Avcil et al. partially agreed with these results and demonstrated that children with ADHD had much higher neutrophil count values than the control group but that their lymphocyte counts were significantly lower (30).

In this study, the inflammation ratio levels in children with ADHD were evaluated by comparing the NLR, PLR, and MLR in pediatric ADHD patients and the control group, and it was found that the NLR, PLR, and MLR values in the ADHD group were significantly higher in ADHD-medicated and unmedicated children than those in the control group. That was confirmed by Akinci and Uzun, who discovered that children and adolescents with ADHD had considerably higher neutrophil, PLR, and NLR numbers than healthy controls, and that these were linked to the low-grade inflammation that is a feature of ADHD (19).

Also, our study showed that the neutrophil/lymphocyte ratio was significantly higher in the unmedicated subgroup than the control group, while there was no significant difference between the medicated and unmedicated subgroups regarding NLR. The monocytes/lymphocytes ratio was significantly higher in the unmedicated subgroup than the medicated ADHD subgroup, which reflects the value of the monocytes/lymphocyte’s ratio as a predictor of ADHD treatment response. This result was agreed with by Önder et al., who showed the significance of NLR is not affected by medication use, age, or sex (31). Regarding the effect of treatment on ADHD in Conner's score, we found that the impulsivity, inattention, and total ADHD scores were significantly higher in the unmedicated group than the medicated group, while there was no significant difference regarding the hyperactivity score between the unmedicated and medicated subgroups.

In our study, we found that there was a significant positive correlation between the ADHD scores and severity and CBC inflammatory ratios (neutrophil/lymphocyte, monocyte/lymphocyte), which may reflect the role of the inflammatory markers in the prediction of ADHD severity, while there was no significant difference between the hyperactivity score and platelets/lymphocyte ratio in all diseased patients.

On the other hand, Fahiem and Mekkaw found no statistically significant relationship between simple blood inflammatory mediators and the various subtypes of ADHD. This suggests that higher inflammatory markers are related to ADHD diagnosis but does not necessarily correspond to the same aspect's severity or seriousness (26).

This study's findings support the theory that inflammation has a significant role in the intricate pathophysiology of ADHD. They also imply that CBC inflammatory ratios, an easily accessible indicator of ADHD severity, are a trustworthy hematological parameter for evaluating the role of inflammation in the etiology of ADHD.

ADHD

Attention deficit hyperactivity disorder

CBC

complete blood count

WBC

white blood cell

MPV

mean platelet volume

NLR

neutrophil lymphocyte ratio

PLR

platelet lymphocyte ratio

MLR

monocyte lymphocytic ratio

IL-1β

Interleukin 1β

IL-6

Interleukin 6

IL-10

Interleukin 10

TNF-α

Tumor necrosis factor alpha

DSM-5

The Diagnostic and Statistical Manual of Mental Illness

EDTA

Ethylene Diamine Tetra acetic Acid

ESR

Erythrocyte Sedimentation Rate

SPSS

Statistical Package for Social Sciences

Ethical Approval

The study had local Research Ethics approval of Minia medical collage ethics committee and Written consent was obtained from patients or their legal guardians.

We confirm that all methods were performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards.

Availability of data and materials

datasets used can be accessed by asking author to provide when needed.

Consent for publication

Not applicable

Competing interest

No conflict of interest with regard to the work.

Funding sources: NO sources for fund, all the cost paid by the authors.

NO financial support or other benefits from commercial sources for the work reported on in the manuscript, No other financial interests of the authors,

Author contribution:

M.F: Selection of research idea, selection of statistics points, review results- sharing in writing.

M.W: Selection of cases- review paper

A.K: Sampling, Laboratory techniques, review paper

Acknowledgement: 'Not applicable'

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Complete Blood Count Inflammatory Ratios in Attention Deficit Hyperactivity Disorder Children

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