Study of CD64 and HLA-DR as Early Detection Markers of Sepsis in Hepatic ICU Patients

• Sepsis is considered the most common cause of death in liver cirrhosis patients.
• This study pioneers in presenting comprehensive findings on the correlation of immune-related markers with sepsis in a cirrhotic cohort.
• The potentiality of these markers as early indicators of early sepsis management in cirrhotic patients had emphasized the broader relevance of the findings in critical care settings.
• Compared to traditional markers like CRP and S. Lactate, CD64 has comparable diagnostic value to distinguish sepsis in cirrhotic patients with the advantageous timely character allowing prompt management for this high risky critical cohort.

Nevertheless, the need for early predictors of the occurrence of sepsis and mortality in patients with liver cirrhosis was only conducted by investigating various immune cell markers, including PMN CD64%, PMN CD64 MFI, PMN HLA-DR%, Mono CD64%, Mono CD64 MFI, Mono HLA-DR MFI, and Sepsis Index CD64, a high-affinity Fcγ receptor, exhibits heightened expression on neutrophils during systemic inflammatory response syndrome [SIRS], making it a promising early marker for bacterial infection [6].

Correspondingly, reduced monocytic HLA-DR (mHLA-DR) expression serves as a consistent marker for immunosuppression in sepsis patients [7].

Flow cytometry (FCM) emerges as a valuable diagnostic tool for immune-related disorders, offering insights into the systemic response to infection through profiling cytokines and surface markers (e.g., neutrophil CD64 and mHLA-DR). This was the impulse of the current study assessing the role of neutrophil CD64 and HLA-DR as markers of early detection of sepsis in liver cirrhosis patients.

PATIENTS/MATERIALS AND METHODS

Patients:

This case-control study was conducted on patients diagnosed with liver cirrhosis, either complicated with sepsis or not. Participants were recruited from outpatients, inpatients, and the Intensive Care Unit [ICU] within the Hepatology and Gastroenterology Department at the National Liver Institute, Menoufia University. Patients with cirrhosis were diagnosed based on clinical, laboratory, and radiological criteria [8].

Sepsis diagnosis in patients was based on clinical suspicion (cultures taken or antibiotics started] along with the fulfillment of SIRS criteria (2 or more of White Cell Count (WCC) >11 or <4, Heart Rate (HR) >90, Respiratory Rate (RR) >20, or temperature >38 or <36°C) [9-11].

Exclusion criteria

Participants meeting any of the following criteria: Patients younger than 18 years, acute pancreatitis, septic shock at the time of enrolment, severe organ failure at the time of enrolment (an immediate requirement for ventilation, vasopressor, or renal replacement therapy), hematological malignancy, recent chemotherapy (within the past 2 weeks), myelodysplastic syndromes, known neutropenia, pregnancy, blood transfusion exceeding 4 units in the past week, oral corticosteroids for >24 hours prior to enrolment, patients with human immunodeficiency virus [HIV] or patients with an ICU length of stay less than 24 Hours or more than 100 days were excluded.

Patients were categorized into the following groups: Group 1: 35 ICU Cirrhotic patients with sepsis meeting inclusion criteria, Group 2: 35 cirrhotic patients without sepsis and Group 3: 30 healthy individuals without a history or clinical evidence of liver disease or any other disease, with negative anti-HCV and HBsAg.

For all patients, the following procedures were conducted: History taking [age, sex, history of blood transfusion, history of any surgery], Clinical examination, Chest X ray, Abdominal sonar, Laboratory investigations, including ( Complete blood count [CBC], CRP, kidney function test, Urine analysis, Anti-HCV and detection of HCV RNA by PCR, HBV serological markers [HBsAg and anti-HBc], Liver profile: alanine aminotransferase [ALT], aspartate aminotransferase [AST], serum albumin, total bilirubin, ascitic fluid analysis for spontaneous bacterial peritonitis (12), and Serial measures of serum lactate.

Sepsis index (based on the combination of two CBC parameters: monocyte distribution width (MDW) and mean monocyte volume (MMV) [14] (The Sepsis Index Score from two monocyte parameters often refers to a method that evaluates sepsis risk using monocyte distribution width (MDW) and monocyte volume distribution width (MVW). SI=MDW×MVW/100.

1. MELD−Na−[0.025×MELD×(140−Na)]+14000) [14], ALBI score (log10 bilirubin×0.66)+(albumin×−0.085) [15].
2. Culture and antibiotic sensitivity testing.
3. Detection of PMN CD64%, PMN CD64 MFI, PMN HLA-DR%, PMN HLA-DR MFI, Mono CD64%, Mono CD 64 MFI, Mono HLA-DR%, and Mono HLA-DR MFI using flow cytometry [16, 17].

Sampling

The antibody used in this study underwent quality control testing through immunofluorescent staining with flow cytometric analysis. The recommended amount of reagent was 5 μL of antibody per test [for a million cells in 100 μL staining volume or per 100 μL of whole blood].

Ethical Approval

The study protocol received approval from the ethical committee of our institution, and all selected patients provided informed consent before enrolment in the study.

Statistical analysis

The data was first coded and verified before its entry. The computer program Statistical Package for Social Sciences (SPSS) (ver.21) Chicago, USA was used for analyzing the collected data and for drawing figures.

Data expressed as mean ± standard deviation and number, percentage. Student-t-test and ANOVA tests were used to determine the significant difference for the numeric variable. Chi.-square was used to determine the significant values for categorical variables. Person’s correlation was used for correlations between groups. P value is considered significant when P value < 0.05 and not significant when P value > 0.05.

RESULTS

All demographic and laboratory criteria are illustrated in table 1. The most common causes of infections in liver cirrhosis patients were mainly spontaneous bacterial peritonitis (SBP) (table 2).

CRP at Cut-off Point: >50 showed AUC: 0.882, Sensitivity: 80.0%, Specificity: 77.14%, with positive predictive value (PPV): 77.8%, and negative predictive value (NPV): 79.4%.

Lactate at Cut-off Point: >3.5 showed AUC: 0.750, Sensitivity: 71.43%, Specificity: 82.86%, PPV: 80.6%, and NPV: 74.4%. Mono CD64% at Cut-off Point: >62.9 showed AUC: 0.676, Sensitivity: 80.0%, Specificity: 60.0%, PPV: 66.7%, and NPV: 75.0%.

Mono CD64 MFI at Cut-off Point >9.5 shoed AUC: 0.659, Sensitivity: 94.29%, Specificity: 37.14%, PPV: 60.0%, and NPV: 86.7%.

Patients with sepsis showed that the combination of ALBI score and S. Lactate had sensitivity, specificity, PPV, NPV, and accuracy of 80%, 80%, 80%, 80%, and 0.831%, respectively (figure 1D).

Combinations of CRP + S. Lactate + Mono CD64% + Mono CD64 MFI revealed AUC: 0.929, Sensitivity: 82.86%, Specificity: 91.43%, PPV: 90.6% and NPV: 84.2% (figure 1).

The immune markers only Mono CD64% and Mono CD64 MFI were significantly elevated in septic patients than others (P=0.01) (table 3).

According to Uni-Varity model, early prediction of sepsis in cirrhotic patients we noticed the following results: (Creatinine >1 with OR 4.231 – Urea >107 with OR 8 – Na ≤136 with OR 7.222 - T.B >3.29 with OR 8 – PMN >68.1 with OR 0.120 – Lymphocytes ≤8.4 with OR 11.625 - Mono CD64% >62.9 with OR 6 - Mono CD64 MFI >9.5 with OR 9.750 – CRP >32.4 with OR 13.500 – Lactate >1.93 wit OR 12.083 - ALBI score >0.01 with OR 6 - MELD (UNOS)>7.66 WITH OR 22 - MELD-Na >10.53 with OR 13.5).

According to Muli-Varity model, early prediction of sepsis in cirrhotic patients showed the following: (CRP >32.4 with OR 67.014, Lactate >1.93 with OR 128.345, ALBI score >0.01 with OR 46.461, MELD [UNOS) >7.66 with OR 21.709, and MELD-Na >10.53 with OR 21.184); this is summarized in table 4.

Exploring the diagnostic markers of early sepsis in cirrhotic patients with CAID had emphasized the significance of cell immune markers in timely detection in the ICU setting [17].

The current study had demarcated significant elevations in ordinary sepsis markers like C-reactive protein (CRP), PMN counts, lactate, and sepsis index (P=0.001) in ICU patients with sepsis. This is the usual picture of a cirrhotic septic patient in the ICU [18]. CRP, being an acute-phase reactant produced by the liver during inflammation, is recognized as a valuable marker, known for its characteristic surge during infection, often rising significantly [18]. Additionally, D'Abrantes. et al, emphasized the predictive value of plasma lactate levels in assessing the prognosis of sepsis [19]. Hyperlactatemia and lactic acidosis, as observed in our study, may result from increased lactate production due to impaired tissue oxygenation, stemming from reduced oxygen delivery or disorders in oxygen utilization, ultimately leading to heightened anaerobic metabolism [20].

In this study, a significantly elevated total bilirubin (TB) in septic patients compared to the non-septic cirrhotic group, pointing to the role of sepsis in this

elevation. Cholestasis-induced sepsis (CIS) differs from hepatic cellular dysfunction-associated cirrhosis in its gradual onset post-ICU admission, marked by rising bilirubin, alkaline phosphatase, and gamma-glutamyl transferase levels.

Cirrhotic patients with sepsis demonstrated elevated ALBI scores, MELD (UNOS), and MELD-Na values in comparison to non-septic cases. This observation aligns with the understanding that sepsis is a significant milestone in the progression towards mortality, and all these scores serve as reliable indicators of short-term survival [21]. Elevated bilirubin due to CIS contributes greatly to these elevations. Also, albumin, which is significantly reduced in cirrhosis-sepsis cases might be an important discriminator [22]. Hypoalbuminemia is more common in sepsis patients, particularly those with septic shock, due to the leakage of protein-rich fluid caused by capillary dysfunction. Previous research indicates that low serum albumin levels are associated with higher mortality risk in sepsis [23-24].

However, the need for early predictors of the occurrence of sepsis and mortality in patients with liver cirrhosis was only conducted by investigating various immune cell markers, including PMN CD64%, PMN CD64 MFI, PMN HLA-DR%, Mono CD64%, Mono CD64 MFI, Mono HLA-DR MFI, and Sepsis Index (SI).

Statistically significant elevations in Mono CD64%, Mono CD64 MFI, and SI were observed in cirrhotic patients, with sepsis, indicating altered immune responses and signifying their prognostic value.

The diagnostic accuracy of these markers was assessed with sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. CRP at Cut-off Point: >50 showed AUC: 0.882, Sensitivity: 80.0%, Specificity: 77.14%, with positive predictive value (PPV): 77.8%, and negative predictive value (NPV): 79.4%. Lactate at Cut-off Point: >3.5 showed AUC: 0.750, Sensitivity: 71.43%, Specificity: 82.86%, PPV: 80.6%, and NPV: 74.4%. Mono CD64% at Cut-off Point: >62.9 showed AUC: 0.676, Sensitivity: 80.0%, Specificity: 60.0%, PPV: 66.7%, and NPV: 75.0%. while Mono CD64 MFI at Cut-off Point >9.5 shoed AUC: 0.659, Sensitivity: 94.29%, Specificity: 37.14%, PPV: 60.0%, and NPV: 86.7%. Patients with sepsis showed that the combination of ALBI score and S. Lactate had sensitivity, specificity, PPV, NPV, and accuracy of 80%, 80%, 80%, 80%, and 0.831%, respectively. Combinations of CRP + S. Lactate + Mono CD64% + Mono CD64 MFI revealed AUC: 0.929, Sensitivity: 82.86%, Specificity: 91.43%, PPV: 90.6%, and NPV: 84.2%. Mono CD64 MFI (94.29%) stands out as having the highest sensitivity, which is crucial for identifying true positive cases. The combined use of CRP, S. Lactate, Mono CD64%, and Mono CD64 MFI revealed the highest AUC (0.929). Additionally, this combination had the highest specificity needed for minimizing false positives (better overall diagnostic performance).

Prior studies by Davis et al., Hsu et al., and Dal Ponte et al. have underscored the superior diagnostic performance of nCD64 over traditional markers like white blood cell count, erythrocyte sedimentation rate, and CRP [25-27]. Icardi et al. further highlighted the predictive value of nCD64 with a sensitivity of 94.6% and a specificity of 88.7% [28]. Lewis et al. and Zhou et al. emphasized the discriminatory power of CD64, particularly in distinguishing septic shock patients [29-30]. Additionally, the combination of nCD64 and CRP has been shown to enhance sepsis diagnosis [31]. Chauhan et al. advocated for flow cytometry analysis of nCD64, asserting its superiority in sepsis detection [32].

In a recent study by Verma et al., the mean fluorescence intensity (MFI) of neutrophil CD64 (nCD64) was markedly elevated in both sepsis and non-sepsis groups compared to controls, demonstrating the diagnostic potential of nCD64 [33].

In summary, the combined use of CRP, S. Lactate, Mono CD64%, and Mono CD64 MFI has a higher AUC and shows promising sensitivity, specificity, PPV, and NPV, making it a potentially effective diagnostic tool for sepsis.SO, healthcare professionals may improve their ability to identify sepsis at an early stage, facilitating prompt and targeted interventions for improved patient outcomes.

 Furthermore, predictive models based on univariate and multivariate analyses highlighted key predictors for early sepsis detection in cirrhotic patients, emphasizing the significance of factors such as creatinine, urea, sodium, total bilirubin, PMN, lymphocytes, Mono CD64%, Mono CD64 MFI, CRP, S. Lactate

In addressing the limitations of this study, such as the sample size and potential confounding factors, we delve into recommendations for future research. It is suggested that future investigations should focus on a more in-depth exploration of specific immune markers and their dynamics in larger-size studies on patients with cirrhosis.

This study is a pioneer in presenting comprehensive findings on the correlation of immune-related markers with sepsis in a cirrhotic cohort, showcasing the potential of these markers as early indicators of early sepsis management in cirrhotic patients, emphasizing the broader relevance of the findings in critical care settings.

CONCLUSION

Conclusively, compared to traditional markers like CRP and S. Lactate, CD64 has comparable diagnostic value for distinguishing sepsis in cirrhotic patients with the advantageous timely character allowing prompt management for this high-risk critical cohort.

Acknowledgments

The authors would like to all colleagues at the National Liver Institute, Menoufia University

Abbreviations

MELD: Model for End-Stage Liver Disease, ICU: intensive care unit, CBC: Complete Blood Count, CRP: C reactive protein, PMN: polymorph nuclear leukocytes, TLC ascitic: total leukocytic count in ascitic, SI: Sepsis index, CXR: chest x-ray, IQR: inter quartile ratio, CAS: cholestasis associating sepsis.

Funding: None

Conflict of Interest: The authors declare no conflict of interest

Author Contributions:

Conceptualization, E.M., KH.F. and A.G.; methodology, A.F., KH.F.; formal analysis, A.M., and A.M.; data curation, A.F., A.M.; writing—original draft preparation, A.F.; writing—review and editing, E.M.; supervision, A.G., A.M.; funding acquisition, A.F. All authors have read and agreed to the published version of the manuscript.

Ethical approval: The Institutional Review Board at National Liver Institute, Menoufia University determined that our study was exempt from review.

Availability of data and materials:

Data available upon request.