White blood cell counts, ratios, and C-reactive protein among individuals with schizophrenia spectrum disorder and associations with long-term outcomes: a population-based study

Background: Immune mechanisms are associated with adverse outcomes in schizophrenia; however, the predictive value of various peripheral immune biomarkers has not been collectively investigated in a large cohort before. Objective: To investigate how white blood cell (WBC) counts, ratios, and C-Reactive Protein (CRP) levels influence the long-term outcomes of individuals with schizophrenia spectrum disorder (SSD). Methods: We identified all adults in the Central Denmark Region during 1994 – 2013 with a measurement of WBC counts and/or CRP at first diagnosis of SSD. WBC ratios were calculated, and both WBC counts and ratios were quartile-categorized (Q4 upper quartile). We followed these individuals from first diagnosis until outcome of interest (death, treatment resistance and psychiatric readmissions), emigration or December 31, 2016, using Cox regression analysis to estimate adjusted hazard ratios (aHRs). Results: Among 6,845 participants, 375 (5.5 %) died, 477 (6.9 %) exhibited treatment resistance, and 1470 (21.5 %) were readmitted during follow-up. Elevated baseline levels of leukocytes, neutrophils, monocytes, LLR, NLR, MLR, and CRP increased the risk of death, whereas higher levels of lymphocytes, platelets, and PLR were associated with lower risk. ROC analysis identified CRP as the strongest predictor for mortality (AUC = 0.84). Moreover, elevated levels of leukocytes, neutrophils, monocytes, LLR, NLR and MLR were associated with treatment resistance. Lastly, higher platelet counts decreased the risk of psychiatric readmissions, while elevated LLR increased this risk. Conclusions: Elevated levels of WBC counts, ratios, and CRP at the initial diagnosis of SSD are associated with mortality, with CRP demonstrating the highest predictive value. Additionally, certain WBC counts and ratios are associated with treatment resistance and psychiatric readmissions.

WBC counts, NLR, and CRP have all shown a significant association with overall mortality in the general population (Fest et al., 2019;Park et al., 2023;Proctor et al., 2015;Song et al., 2021).They have also been linked to outcomes and can predict disease progression in patients with various medical conditions (Guo and Zou, 2023;de Jager et al., 2010;Liu et al., 2020;Ni et al., 2020;Park et al., 2018;Templeton et al., 2014).In patients with mental disorders treated in secondary mental health services, elevated NLR has been associated with increased mortality but not with hospital admissions, as reported in a cross-sectional study without specific information about disorder-specific mortality (Brinn and Stone, 2020).Additionally, in individuals with schizophrenia, higher levels of CRP have been linked to increased mortality (Horsdal et al., 2017a), and various immune system markers may affect treatment response (Fond et al., 2020;Kose et al., 2021;Llorca-Bofí et al., 2024a;Orbe and Benros, 2023).However, to our knowledge, there is a lack of large-scale longitudinal studies that investigate different inflammatory biomarkers and long-term outcomes among patients with schizophrenia.Therefore, it is crucial to examine the association between WBC counts, ratios, and CRP assessed at the time of diagnosis and subsequent prognosis in schizophrenia (Bhikram and Sandor, 2022).Such investigations may provide valuable insights for developing prediction models suitable for routine use and for enabling more personalized treatment early in the course of the disorder.This is particularly important because individuals with schizophrenia experience an 11-13-year shorter life expectancy (Laursen et al., 2019;Llorca-Bofí et al., 2024c).
In this first large-scale population-based study of inflammatory biomarkers at first diagnosis of schizophrenia spectrum disorder (SSD), we investigated the associations between WBC counts and ratios, as well as CRP, with three long-term outcomes: 1) all-cause mortality, 2) treatment resistance, and 3) psychiatric readmissions.

Registers
This study was based on the linking of Danish health care registries and databases covering the entire Danish population by using the unique civil registration number assigned to every Danish citizen in The Danish Civil Registration Register since 1968 (Pedersen, 2011).For a more detailed description of the registers see Supplementary Material.

Study population
Diagnoses of SSD were obtained from The Danish Psychiatric Central Research Register.The diagnoses are coded according to the International Classification of Disease (8th revision  until the end of 1993, and 10th revision [ICD-10] thereafter).We identified individuals with a first main diagnosis of SSD (see Table S1 for ICD codes) in an in-or outpatient setting (index date being first admission date for inpatients or first contact date for outpatients) between January 1, 1994, and December 30, 2013.The final study population included individuals who were born in Denmark after January 1, 1955, aged 18 years or older at the diagnosis, and were living in Central Denmark Region at the time of diagnosis identified by linkage to The Danish Civil Registration System.A flowchart illustrating the inclusion and exclusion criteria is shown in Figure S1.

Exposure: assessment of WBC counts, ratios and CRP
We used the LABKA research database (Grann et al., 2011) to identify all measurements for leukocyte count, neutrophil count, monocyte count, platelet count, lymphocyte count and CRP within ± 30 days from index date and used the measurement closest to index date as the baseline measurement (see Table S2 for blood component codes).We computed WBC ratios as follows: leukocyte to lymphocyte counts ratio for LLR; neutrophil to lymphocyte counts ratio for NLR; monocyte to lymphocyte counts ratio for MLR; platelet to lymphocyte counts ratio for PLR.As there are no currently defined cut-off values for WBC ratios in psychiatric patients (Bhikram and Sandor, 2022), we divided them into four groups based on quartiles in our dataset: Q1 (lower quartile), Q2, Q3, and Q4 (upper quartile) at baseline.A composite index was defined as having ≥ 2 indexes in Q3 or Q4.We adhered to the recommendations outlined by the U.S. Centers for Disease Control and Prevention and the American Heart Association (Myers et al., 2004) to classify CRP levels at baseline into the following groups: low level of systemic inflammation (<1 mg/L); intermediate level of systemic inflammation (1-2.9 mg/L); high level of systemic inflammation (3-10 mg/L); and acute inflammation (>10 mg/L).In the analyses, we employed a time-dependent exposure approach.Measurements of WBC counts, ratios and CRP taken later than 30 days after baseline were grouped according to the cut-off at baseline and included in the analysis from the time of their measurement.Therefore, patients with additional assessments during follow-up had their measurements updated accordingly during the follow-up period.

Outcomes: assessment of all-cause mortality, psychiatric readmissions, and treatment resistance
Three outcomes where defined: a) All-cause mortality: it was ascertained from The Danish Civil Registration System (Pedersen, 2011).b) Treatment resistance: we identified any subsequent use of clozapine or electroconvulsive therapy (ECT) that occurred more than 14 days after the first contact (discharge date for inpatients and contact date for outpatients).We used The Danish National Prescription Registry (Wallach Kildemoes et al., 2011) to identify clozapine prescription, the LABKA (Grann et al., 2011) research database to identify blood test for clozapine levels, and The Danish National Patient Registry to identify ECT procedures (Table S3 for clozapine and ECT codes).c) Psychiatric readmissions: we identified any subsequent admission in an inpatient setting at a psychiatric hospital that occurred more than 14 days after the first contact (discharge date for inpatients and contact date for outpatients) using data from The Danish Psychiatric Central Research Register (Mors et al., 2011).

Assessment of baseline characteristics
We obtained information from multiple Danish national registers: The Danish National Patient Register was used to sum the number of contacts (in-and outpatient and emergency room contacts) at the somatic hospitals within 1 year prior to index date, including the number of days hospitalized (below or above 10 days).We also assessed cardiovascular risk factors defined as the presence of hypertension, diabetes mellitus, dyslipidemia, and/or obesity (see Table S4 for ICD codes).Finally, we identified individuals with autoimmune disorders or severe infections diagnosed within one month prior to the blood test and onwards (see Table S5-S6 for ICD codes).
The Psychiatric Central Research Register was used to assess comorbid substance use disorders (see Table S4 for ICD codes).
The Danish Education Registers (Jensen and Rasmussen, 2011) were used to assess highest completed education level (primary school vs. a combined group of higher-level education. The Danish National Prescription Registry (Wallach Kildemoes et al., 2011) was used to trace anti-inflammatory prescriptions (anti-inflammatory drugs, corticosteroids, and analgesics) filled in the month before the index date (see Table S4 for anti-inflammatory ATC-codes).

Statistical analysis
Baseline characteristics, including WBC counts, ratios, and CRP levels among different SSDs, were analysed.Categorical variables were compared using Chi-square tests, while continuous variables were assessed using Kruskal-Wallis tests (Wilcoxon rank-sum test).Regarding the risk of the outcome, follow-up began on the index date or the date of the alternate LABKA measure and ended on the date of inpatient admission, treatment resistance (incident use of clozapine or ECT), death, emigration, or December 31, 2016.For individuals hospitalized at their first diagnosis who were only at risk of admission after discharge, their follow-up began on the date of discharge when examining admissions.We utilized Cox proportional hazards regression analysis to compute hazard ratios (HRs).Three adjusted models were used sequentially in an additive manner.The first model included gender, age, and calendar period.The second model added family history of psychiatric disorders and education level of the parents.The third, or fully adjusted model, added cardiovascular risk factors, comorbid substance use disorder, and prior use of anti-inflammatory drugs.The fully adjusted model was used for the reported data except when specified otherwise.Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of WBC counts, ratios and CRP for mortality.Statistical analyses were conducted using Stata version 13.1 (StataCorp LP, College Station, Texas, USA), and ROC analysis was performed using the pROC package in R (Robin et al., 2011).The statistical significance level was set at 0.05 in all analyses.

Sensitivity analysis
We carried out two sensitivity analyses.First, to reduce misdiagnosis with other psychotic disorders, we restricted analyses to patients with schizophrenia (ICD-10: F20).Secondly, to minimize confounding factors that could influence measured biomarkers at the time of the blood test, we restricted the analysis by excluding patients diagnosed with autoimmune diseases or severe infections within one month prior to the blood test and onwards.

Population characteristics
During the study period, a total of 33.887 individuals with incident SSD were identified.Among all individuals, a total of 6.845 (20.1 %) had a first-time SSD diagnosis of schizophrenia (n = 2.636) or other psychotic disorders (n = 4.209) and a complete WBC counts and/or CRP measurement at baseline.Baseline characteristics among individuals with SSD and baseline measurements are shown in Table 1.Individuals diagnosed with schizophrenia differed from those diagnosed with other psychotic disorders in terms of gender, age at the initial diagnosis, inpatient status at the initial diagnosis, number of somatic and psychiatric contacts in the previous year, use of clozapine, and use of antiinflammatory treatment in the preceding month (all P<0.05).

Table 1
Baseline characteristics for individuals with schizophrenia spectrum disorder included in the study.

All-cause mortality
Among the 6.845 individuals with WBC counts and/or CRP measured at baseline, 375 (5.5 %) died during the follow-up period.Rates and hazard ratios (HRs) of all-cause mortality for individuals with SSD are shown in Table 2, Table S17 and Fig. 1.

Treatment resistance
Among the 6.845 individuals with WBC counts and/or CRP measured at baseline, 477 (6.9 %) exhibited treatment resistance during the follow-up period.Rates and HRs of treatment resistance for individuals with SSD are shown in Table 2 and Table S18.

Psychiatric readmissions
Among the 6.845 individuals with WBC counts and/or CRP measured at baseline, 1470 (21.5 %) were admitted to a psychiatric hospital during the follow-up period.Rates and HRs of psychiatric readmissions for individuals with SSD are shown in Table 2 and  Table s19.
Individuals with baseline Q4 levels of platelet counts had a lower risk of psychiatric readmissions, with an aHR of 0.76 (95 % CI: 0.63-0.90).In contrast, LLR in Q4 increased the risk of psychiatric readmission, with an aHR of 1.32 (95 % CI: 1.08-1.61).Non-significant associations were observed for the other WBC counts, ratios, or CRP levels.
When conducting sensitivity analyses for individuals with autoimmune disease or severe infection, we excluded/censored a small number of cases, which prevented us from reporting specific findings regarding this subgroup.

Discussion
We conducted the largest longitudinal population-based study to date, assessing WBC counts, ratios, and CRP at the time of the first diagnosis of SSD, and investigated their associations with all-cause mortality, treatment resistance, and psychiatric readmissions.Our findings were as follows: (1) higher baseline leukocytes, neutrophils, monocytes, LLR, NLR, MLR, and CRP were associated with increased allcause mortality, while lymphocytes, platelets, and PLR decreased the risk; (2) CRP demonstrated the highest predictive value for mortality with a ROC-AUC of 0.84; (3) elevated levels of leukocytes, neutrophils, monocytes, LLR, NLR, and MLR were associated with treatment resistance; (4) higher platelets decreased the risk of psychiatric readmissions, while LLR increased this risk.Our results underscore the need for a greater focus on somatic states among individuals with SSD to prevent premature mortality and highlight the potential of WBC counts, ratios, and CRP in assessing longitudinal prognosis in this population.
Inflammatory activation is associated with higher mortality in the general population (Proctor et al., 2015).Some markers of the inflammatory response have demonstrated independent prognostic value in patients with a variety of medical conditions.In SSD, there is a higher risk of premature mortality compared to the general population, mainly due to cardiovascular causes (Plana-Ripoll et al., 2022).We previously demonstrated that higher CRP levels at first diagnosis of schizophrenia are associated with increased mortality, particularly among individuals with somatic comorbidity (Horsdal et al., 2017a).In this new study, we analysed WBC counts and ratios and found that leukocytes, neutrophils, monocytes, LLR, NLR and MLR are also associated with all-cause mortality.These findings align with previous studies using WBC counts and ratios in the general population (Abete et al., 2019;Chan et al., 2022;Fest et al., 2019;Park et al., 2023;Shah et al., 2017;(Song et al., 2021)) and other medical conditions (de Jager et al., 2010;Lattanzi et al., 2019;Templeton et al., 2014).Additionally, we evaluated the predictive abilities of WBC counts, ratios and CRP for mortality.Our findings indicate that CRP exhibits the highest predictive accuracy for mortality, with an AUC of 0.84.Consequently, we affirm the independent association between inflammation and mortality and highlight CRP as a predictor when assessing this relationship in SSD.Interestingly, we observed that lymphocytes, platelets, and PLR were associated with a lower risk of mortality.There are inconsistencies in the literature Bold values denote statistical significance.
regarding their predictive value.Some studies report that higher lymphocyte counts are linked to increased mortality (Abete et al., 2019), while others suggest a connection with lower lymphocyte counts (Izaks et al., 2003).Similarly, the association between higher platelet counts and mortality has been noted in some studies within the general population, but not all studies replicate this finding (Izzi et al., 2018).However, our findings are novel because these associations have not been previously investigated in schizophrenia, suggesting a need for further investigation in the field.Treatment resistance is not a homogeneous subgroup within SSD, and different clinical and neurobiological pathways may be involved in the condition (Potkin et al., 2020).Hypotheses suggest that inflammation at an early age followed by chronic low-grade activation contributes to treatment resistance (Ioannou et al., 2021).Osimo and collaborators showed that elevated monocyte and lymphocyte counts at FEP were linked to poor clinical outcomes, defined as continued involvement in secondary mental health services, even after adjusting for four confounders (Osimo et al., 2021b).Additionally, they developed a machine-learning model to forecast the 8-year likelihood of treatment resistance (defined by clozapine use) starting from FEP.This model integrated various analytical including neutrophils and lymphocytes, achieving an AUC of approximately 0.67 (Osimo et al., 2023).In our study, lymphocytes were not associated with treatment resistance, but we found associations with a high number of immune cells and ratios (leukocytes, neutrophils, monocytes, LLR, NLR, and MLR), reinforcing the idea of an involvement of the immune system in treatment resistance.The observed differences in lymphocyte levels might be due to varying distributions of inflammatory markers among ethnic groups and the impact of comorbidities (Coates et al., 2020;Lang et al., 2021).A combined measure of inflammatory markers has been suggested to better explain these findings and WBC ratios may be a good choice to detect imbalances between the innate and adaptive immune pathways.In this line, Bioque and collaborators found that in FEP psychosis patients, those who did not meet remission criteria at the two-year followup had significantly higher NLR than the group in remission (Bioque et al., 2022).Our results align with this, indicating that not only NLR,  Abbreviations: LLR=Leukocyte-to-Lymphocyte ratio; NLR=Neutrophil-to-lymphocyte ratio; PLR=Platelet-to-Lymphocyte ratio; MLR=Monocyte-to-Lymphocyte ratio; CRP=C-Reactive Protein; AUC=Area Under the Curve.
but also LLR and MLR, are associated with long-term treatment resistance.In terms of CRP, Nettis and collaborators found that higher CRP in combination with other metabolic parameters were associated with poor clinical response at 1-year follow-up after a FEP (Nettis et al., 2019), and Fond and collaborators showed that CRP levels were associated with ultra-resistant schizophrenia (Fond et al., 2019).However, these findings were not replicated in our group when we analysed first-episode schizophrenia patients who later developed treatment resistance (Horsdal et al., 2017b).In the current study, which includes a larger sample size, we again found no association, replicating our previous results.
Several factors have been reported to contribute to readmission in SSD (Mi et al., 2020).However, no single inflammatory biomarker has demonstrated a consistent association with psychiatric readmissions (Brinn and Stone, 2020;(Horsdal et al., 2017a,b;Llorca-Bofí et al., 2024)).Surprisingly, in our study, higher platelet levels decreased the risk of psychiatric readmissions, while lymphocyte-to-leukocyte ratio (LLR) increased this risk in the whole SSD population; however, these results were not maintained when analysing schizophrenia patients only.Our results on platelet associations contrast with those reported by Osimo and collaborators (2021b), who found that higher platelet values were associated with worse psychiatric outcomes in a cohort of FEP patients, although this association was lost after adjusting for confounders.In contrast, our study shows that higher platelet levels lower the risk of psychiatric readmission.This discrepancy may be due to different study populations (established SSD vs. FEP) and outcome definitions (readmission vs. involvement in secondary mental health services).Future investigations should replicate these results and further explore the role of platelets in SSD (Asor and Ben-Shachar, 2012).Finally, LLR has not been previously investigated in relation to longitudinal outcomes in SSD (Mazza et al., 2020), and therefore, this result has not been previously reported.It is essential to replicate this association, as it may be a chance finding.

Strengths and limitations
A major strength of this study is its size and comprehensiveness: it is a population-based register study of up to 22 years' follow-up of all adult and incident SSD patients in a whole region of Denmark, with very little missing data.The use of these registries minimized the risk of both selection and information bias with a high validity of the diagnoses used to identify the study population (Uggerby et al., 2013).Another advantage is the ability to adjust for a range of possible confounding variables collected independently of the blood tests through linking Danish health care registries and databases (Jensen and Rasmussen, 2011;Lynge et al., 2011;Mors et al., 2011;Pedersen, 2011;Petersson et al., 2011;Wallach Kildemoes et al., 2011).Some limitations should be considered when interpreting our results.First, a potential weakness of our study is that it was not possible to include a healthy control group since everyone in the clinical laboratory information system had been in contact with a hospital or general practitioner due to suspected illness.Second, we only included individuals who had a WBC counts and/or CRP measurement within ± 30 days of the initial SSD diagnosis, whereby we may have introduced selection bias as there might be indications for the blood testing beyond monitoring in SSD, thus limiting generalizability.To further understand the potential impact of selection bias, we explored the differences between patients included and not included in the study (Table S21).We found higher proportions of female patients, individuals with higher education levels, as well as higher somatic and psychiatric contacts in the previous year among those included.No differences were found in terms of age, cardiovascular risk factors, or diagnosis.Third, due to the lack of information on symptom severity, we could not use the TRIPP criteria for the definition of treatment resistance (Howes et al., 2017).Notwithstanding, we defined treatment resistance as the use of clozapine and/or ECT, which are two of the strategies proposed by the TRIPP consensus for this type of patient (Kane et al., 2019).Fourth, due to the register-based origin of the blood sample data, factors related with the blood sampling that are known to modify immune cells such as previous fasting, sleep disturbance or timing of the procedure were not available (Sandberg et al., 2021;Villar et al., 2023).Fifth, blood tests were not analysed at the same laboratory.Therefore, despite regular quality control and calibration for high precision in all laboratories, potential inter-laboratory differences in the measurements cannot be ruled out.Finally, we did not have information on factors such as smoking, weight, nutrition, and physical activity, which can influence inflammatory processes in the body (Howard et al., 2019).

Conclusions
Elevated levels of WBC counts, ratios, and CRP at the first diagnosis of SSD increased the risk of long-term mortality, except for lymphocytes, platelets, and PLR, which reduce the risk.CRP exhibited the highest predictive value for mortality.Treatment resistance was associated with elevated levels of leukocytes, neutrophils, monocytes, LLR, NLR, and MLR.Higher levels of platelets decreased the risk of psychiatric readmissions, while LLR increased this risk.Consequently, WBC counts, ratios, and CRP at the first diagnosis of SSD demonstrated long-term predictive potential.

inflammatory drugs, corticosteroids or analgesics the previous month and
onwards, no (%)

Table 2
Associations of baseline WBC counts, ratios, and CRP with mortality, treatment resistance, and readmissions in schizophrenia spectrum disorder.

Table 3
Receiver-operating characteristic (ROC) curve of WBC counts, ratios and CRP for mortality in schizophrenia spectrum disorder.