Early and Late Neutrophil CD64 and Monocyte HLA-DR as Biomarkers for Septic Shock

Detailed Protocol of the Study

• Title Early and late neutrophil CD64 and monocyte HLA-DR as biomarkers for septic shock.
• Study Design

• Type: Prospective, single-center, observational cohort study.
• Setting: Medical ICU of a tertiary care university teaching hospital in northern India.
• Duration: January 2019 - December 2019.
• Ethics Approval: Biomedical Research and Ethics Committee (BREC/18/194).
• Guidelines: Conducted in adherence with STROBE reporting standards.
• Objectives

• Primary Objective: To evaluate neutrophil CD64 (nCD64), monocyte HLA-DR (mHLA-DR), and the Sepsis Index as prognostic markers in septic shock.
• Secondary Objectives:
• Compare biomarker levels between survivors and non-survivors.
• Assess predictive accuracy of these markers for 28-day mortality using ROC analysis.
• Study Population

• Inclusion Criteria:
• Age ≥18 years.
• Diagnosis of septic shock according to prevailing Sepsis-3 criteria.
• Written informed consent from patient or next of kin.
• Exclusion Criteria:
• Pregnant females.
• Active malignancy.
• Severe chronic liver disease.
• Chronic kidney disease requiring maintenance hemodialysis.
• Immunocompromised patients or those on immunomodulatory therapy prior to sepsis onset.
• Non-bacterial sepsis (viral, fungal, tubercular).
• Enrollment & Sample Size

• Screened: 80 patients.
• Excluded: 24 (per exclusion criteria).
• Final cohort: 56 patients (37 survivors, 19 non-survivors).
• Controls: 20 healthy volunteers for baseline biomarker values.
• Data Collection

• Demographics: Age, sex, residence, BMI, smoking status.
• Clinical variables: Primary diagnosis, comorbidities, severity scores (qSOFA, SIRS, APACHE II), vital signs, organ dysfunction parameters (PaO₂/FiO₂, inotropic support, renal replacement therapy, GCS).
• Laboratory parameters: CBC, renal/liver function tests, ABG, primary diagnosis details.
• Outcome measures: ICU stay, ventilator days, 28-day mortality.
• Biomarker Assessment

• Markers:
• Neutrophil CD64 (nCD64).
• Monocyte HLA-DR (mHLA-DR).
• Sepsis Index = nCD64 ÷ mHLA-DR.
• Method: Flow cytometry (8-color FACS Canto II, BD Biosciences).
• Units: Mean Fluorescence Intensity (MFI).
• Time points:
• Day 1 (admission).
• Day 8 (follow-up).
• Statistical Analysis

• Software: SPSS v24 (IBM).
• Descriptive statistics: Mean ± SD for normal data; median (IQR) for skewed data.
• Comparisons:
• Independent t-test for normal distribution.
• Mann-Whitney U test for skewed data.
• Predictive analysis: ROC curves for nCD64, HLA-DR, and Sepsis Index.
• Significance threshold: p < 0.05 (two-sided).
• Outcomes

• Primary outcome: 28-day mortality.
• Secondary outcomes: ICU stay duration, ventilator days, organ dysfunction severity.
• Safety & Compliance

• Biomedical waste: Managed per Biomedical Waste (Management and Handling) Rules.
• Patient safety: Standard ICU protocols followed; no intervention beyond routine care.

Inclusion criteria

Patients were eligible if they met all of the following:

• Age ≥ 18 years.
• Diagnosis of septic shock according to prevailing Sepsis-3 criteria at the time of study.
• Admission to the medical ICU of the tertiary care teaching hospital.
• Written informed consent obtained from patient or next of kin.

Exclusion criteria

Patients were excluded if they met any of the following:

• Pregnant females.
• Active malignancy (any known cancer diagnosis).
• Severe chronic liver disease.
• Chronic kidney disease requiring maintenance hemodialysis.
• Immunocompromised state, including:
• HIV/AIDS.
• Long-term corticosteroid therapy.
• Other immunosuppressive conditions.
• Prior immunomodulatory treatment before onset of sepsis (e.g., biologics, GM-CSF, interferons).
• Non-bacterial sepsis, including:
• Viral sepsis.
• Fungal sepsis.
• Tubercular sepsis.