Measurement of inflammatory markers and lymphocyte subsets
Blood was collected on median days of 6 (2-8) for the hospitalized patients and 7.5 (6.75-8.25) for the non-hospitalized group from the onset of symptoms. Blood was sent for complete blood count (CBC), renal function tests (RFT), liver function tests (LFT), lactate dehydrogenase (LDH), ferritin, D-dimer, coagulation profile, IL-6, and lymphocyte subsets analysis. IL-6 concentration was measured on serum samples using fully automated Elecsys IL-6 immunoassay (electrochemiluminescence immunoassay) on Cobas e 601 immunoassay analyzers (Roche Diagnostics, Switzerland) following the manufacturer’s protocol. The used cut-off of IL-6 was 7.0 pg/ml.
Assessment of different basic lymphocyte and detailed T cell subsets using flow cytometry was performed. DuraClone IM T cell subsets tube (Beckman coulter) that includes CD45RA (clone, 2H4), CD197 (CCR7) (clone, G043H7), CD28 (clone, CD28.2), CD279 (PD1) (clone, PD1.3.5), CD27(clone, 1A4.CD27), CD4(clone, 13B8.2), CD8(clone, B9.11), CD3(clone, UCHT-1), CD57(clone, NC1), CD45(clone, J33) was used to assess different T cell subsets. DuraClone IM phenotyping basic tube (Beckman coulter) that includes: CD16(3G8), CD56(N901), CD19(J3_119), CD14(RMO52), CD4 (13B8.2), CD8(B9.11), CD3(UCHT-1), CD45(J33) was used for basic lymphocyte staining. Gating strategies are presented in figure1 and 2. A total of 100,000 event were collected.
Briefly, 100 µl of blood was added to the tube containing the desired cocktail of antibodies and incubated for 20 minutes at room temperature. 100 µl of lysing solution optilyse-B or Versalyse was added according to the manufacture’s recommendation. This was followed up with a wash step. Acquisition of samples was done using Navios flow cytometry (Beckman coulter) and analyzed using Kaluza version 2.1(Beckman coulter).