Leukocyte Counts, Differential

Anticoagulated whole blood from routine controls were processed on Sysmex XE2100 [TOA Medical Electronics, Kobe, Japan], Normal controls were analysed on Sysmex XN2000 (TOA Medical electronics Co, Kobe, Japan), Advia 2120 (Bayer Diagnostics, Tarritown, NY, USA), DXH800 (Beckman Coulter, Miami, FL USA), Cell-Dyn Sapphire (Abbott Diagnostics Santa Clara, CA, USA) for the determination of the complete blood cell counts and differential counts of leukocytes. The absolute neutrophil count was divided by the absolute lymphocyte count to calculate the NLR.

Demographic, clinical and laboratory data pertaining to both COVID-19 and RMD were recorded for all included patients according to a predesigned proforma. Clinical symptoms attributable to COVID-19, vital signs, treatment(s) administered for COVID-19 (antivirals and/or immunomodulatory therapies) and the final outcome (death or recovery) were noted. Severe and critical disease as defined by the World Health Organization (WHO) were grouped together as severe disease for the purpose of the present study and defined as the presence of oxygen saturation <90% on room air or signs of severe respiratory distress in addition to signs of pneumonia or presence of ARDS, sepsis, septic shock or other conditions that would normally require the provision of life-sustaining therapies, such as mechanical ventilation (invasive or non-invasive) or vasopressor therapy [18 ]. In addition, based on national guidelines [19 ], non-severe disease was categorized further as moderate disease (defined by a respiratory rate >24/min or a peripheral oxygen saturation between 90 and 94%) and as mild disease when patients with RT-PCR-confirmed SARS-CoV-2 had only upper respiratory tract symptoms without any hypoxaemia or tachypnoea. Laboratory parameters recorded included haemogram, liver function tests (total and direct bilirubin, aspartate transaminase, alanine transaminase, γ-glutamyl transferase, alkaline phosphatase and serum albumin), renal function tests, CRP, ferritin, D-dimer, fibrinogen, international normalized ratio and procalcitonin (hospitalized patients only). The neutrophil-to-lymphocyte ratio was also calculated from the differential leucocyte count. Data pertaining to the type of underlying RMD, disease activity status (active vs remission) at the time of acquisition of SARS-CoV-2, and ongoing treatment [supportive, NSAIDs, glucocorticoids (GCs), conventional synthetic DMARDs (including HCQ, MTX, LEF, SSZ, AZA, MMF and CYC), biologic DMARDs (rituximab or anti TNF), antifibrotics (nintedanib or pirfenidone) or IVIG] were also noted, along with details of underlying medical co-morbidities. DMARDs were also categorized into immunomodulators (MTX, LEF and SSZ) and immunosuppressants (CYC, AZA, MMF and biologic DMARDs) for the purpose of predictor analysis.
Outcomes of interest included the proportion of patients with severe COVID-19, mortality (as a percentage), hospitalization (as a percentage), intensive care unit (ICU) stay (as a percentage), the requirement for respiratory support (as a percentage) and its level (oxygen delivered by face mask or nasal prongs, non-rebreathing mask, high-flow nasal cannula, non-invasive ventilation or invasive mechanical ventilation). Predictors of COVID-19 severity, mortality and hospitalization were determined.

The HemoCue system (Glucose 201+, Hb201, and WBC DIFF, HemoCue AB, Angelholm, Sweden) was used to determine blood glucose concentration, haemoglobin, total and differential leukocyte counts (including neutrophils, lymphocytes and monocytes) in duplicate from whole blood samples. The coefficient of variation (CV) for blood glucose concentration, haemoglobin and leukocyte counts was 5.1%, 1.6%, and 13.6% respectively. Haematocrit was determined by capillary method in triplicate from heparin whole blood samples using a microhematocrit reader (CV: 0.7%) (Thermo Fisher Scientific). The haemoglobin and haematocrit values were used to determine changes in plasma volume (P_v) relative to baseline, and to correct plasma variables (Dill and Costill, 1974 (link)). Bacterially-stimulated elastase release was determined as previously described (Costa et al., 2009 (link); Costa et al., 2011 (link); Costa et al., 2019a (link); Costa R. J. S. et al., 2020 (link)). The remaining whole blood in the heparin and K3EDTA vacutainers was centrifuged at 4,000 rpm (1,500 g) for 10 min within 15 min of sample collection and aspirated into 1.5 ml micro-storage tubes and frozen at −80°C until analysis. Prior to freezing, two 50 µl aliquots of heparin plasma were used to determine plasma osmolality (P_Osmol), in duplicate (CV: 0.7%), by freeze point osmometry (Osmomat 030, Gonotec, Berlin, Germany). Circulating concentrations of cortisol (DKO001; DiaMetra, Italy), PMN elastase (BMS269; Affymetrix EBioscience, Vienna, Austria), I-FABP (HK406; Hycult Biotech, Uden, Netherlands), sCD14 (HK320; Hycult Biotech), and LBP (HK315, Hycult Biotech) were determined by ELISA. Additionally, systemic cytokine profile [i.e., plasma IL-1β, TNF-α, IL-10, and IL-1ra concentrations] (HCYTMAG-60K, EMD Millipore, Darmstadt, Germany) were determined by multiplex system (Magpix, Luminex, Austin, TX, United States). All variables were analysed as per manufacturer’s instructions on the same day, with standards and controls on each plate, and sample from each participant assayed on the same plate. The intra- and inter-assay CV for analysed biomarkers, respectively, was 6.1% and 10.4% for cortisol, 2.8% and 3.6% for I-FABP, 4.0% and 9.3% for LBP, 3.3% and 4.2% for sCD14, 5.5% and 9.7% for elastase, 16.0% and 16.6% for IL-1β, 14.9% and 15.5% for TNFα, 15.8% and 9.1% for IL-6, 14.7% and 12.6% for IL-8, 15.9% and 11.1% for IL-10, and 9.2% and 8.8% for IL-1ra.