Tissue preparation:
Bone marrow trephine biopsies were done in selected cases. The core specimens were placed in 10% Phosphate Buffered Formalin (PBF). The fixed tissues were processed using automated tissue process of the Neurology lab unit of King Abdelaziz university. The paraffin blocks were cut with a rotary microtome at 3-5 µm thickness. The sections were floated on a warm water bath at 40°C and taken on a glass slide and incubated for 24 hours. The sections were allowed to cool and stained with Hematoxylin and Eosin (H&E) to assess the structural changes under the light microscope.
For immunohistochemical staining, serial paraffin sections were chosen to be stained by 2 markers for detection of the monocytes. The slides were fixed, deparaffinized and hydrated, treated with 0.5% hydrogen peroxide in methanol for 10 min to block endogenous peroxidases, and washed in tap water. Sections were incubated with 10 mM citrate buffer, pH 6.5, and heated in a microwave oven for two cycles of approximately 2 min each for antigen retrieval. After cooling, sections were washed in PBS (pH 7.6) for 5 min. and placed in normal saline. Sections were incubated with anti CD14 mouse primary antibodies for 2 h from (Novocastra, France). The binding of anti-CD14 mouse antibody was revealed by biotinylated goat anti-rabbit and anti-mouse antibody (AbCys, Paris, France), followed by streptavidin–peroxidase complex (AbCys, Paris, France). The peroxidase activity was developed by using diaminobenzidine (DakoCytomation, Trappes, France). Other selected sections were incubated with anti-CD68 (PG-M1) (DAKO, Denmark) mouse antibody for 1 h. The binding of anti-CD68 antibody was revealed by rabbit anti-mouse antibody, followed by incubation with 1 mg/ml DAB solution (3,3tetrahydrochloride diaminobenzidine, Sigma, USA) for 5 min. Anti CD68 is specific for staining the macrophages developed from the monocytes. The slides were counterstained with Mayer’s hematoxylin and mounted in DPX. Then the sections were completely scanned, examined and photographed.
Blood samples:
Immunophenotyping studies were done on the fresh blood samples of patients with ischemic stroke after diagnosis was confirmed by medical consultants. The peripheral whole blood samples (5 ml) is collected in Ethylenediaminetetraacetic acid (EDTA) tubes. (Becton Dickinson, San Jose, USA), ethical approval No 53217. Full blood counts were performed using a Sysmex XS whole blood analyzer (Sysmex Corporation, Kobe, Japan). Immunophenotyping studies were performed on a total of 12 newly diagnosed with ischemic stroke patients from 2020 (King abduaziz university hospital).
Immunophenotyping
Fluorochrome-conjugated CD markers are purchased from Bio Legend, USA. A combination of peridinin chlorophyll protein (PerCP), fluorescein isothiocyanate (FITC), allophycocyanin (APC), and phycoerythrin (PE) conjugated antibodies were selected. CD16- FITC (monocytes), CD14- PerCP/Cy5.5 (monocytes), CD202b (Tie2) and CD45-APC (leukocyte) were used for leukocyte immunophenotyping (surface staining) using the FACS Canto™ II (Table 1).
Table 1: List of antibodies used for flowcytometry
Materials
|
Catalogue number
|
Supplier
|
PE anti-human CD202b (Tie2) Antibody
|
334206
|
BioLegend (San Diego, USA)
|
PerCP/Cy5.5 anti-human CD14 Antihody
|
367110
|
BioLegend (San Diego, USA)
|
FITC anti-human CD16 Antibody
|
360716
|
BioLegend (San Diego, USA)
|
APC anti-human CD45 Antibody
|
20021658
|
Dako (CA, USA)
|
Flowcytometer Calibration
BD FACS Diva CS&T IVD Beads
BD FACS Diva CS&T beads were used to set up the cytometer, to perform daily performance quality control (QC), and to determine lyse/wash (LW) application settings, allow the software to automatically characterize, track, and report measurements of the cytometer. Automated algorithms in the software defined the cytometer baseline. Once baseline mean fluorescence intensity (MFI) target values were defined, the beads were used to run daily performance checks. BD FACS Diva CS&T IVD beads are also used to reset MFI target values when switching to a new lot of beads. In addition, BD FACS Diva CS&T IVD beads were used to manually determine LW application setting LW once saved, application settings were automatically cytometer after the user performs the dieter performance check the instrument performance, base once that day.
Fluorescence compensation
Using BD™ Comp Beads Set Anti-Mouse Ig, κ are polystyrene microparticles which are used to optimize fluorescence compensation settings for multicolor flow cytometric analyses. The set provides two populations of microparticles, the BD™ Comp Beads Anti-Mouse Ig, κ particles, which bind any mouse κ light chain-bearing immunoglobulin, and the BD™ Comp Beads Negative Control, which has no binding capacity. Four tubes each containing 100 μl of staining buffer BSA , were stained with CD16 -FITC, CD202-PE, CD14-PerCP and CD45-APC (positive control) and one tube containing 1 x 106 white blood cells were subjected to sample processing, with equivalent volumes of PBS in place of the CD markers (unstained cells) for electronic optimization. One drop of BD™ Comp Beads Anti-Mouse Ig, κ was added to positive control tubes and one drop of Comp Beads Negative Control was added to unstained tube (negative control). All incubations were performed at ambient temperature, unless otherwise stated. Cells were stained with CD markers for 15 minutes, in the dark. Unstained tube was then incubated in 2 mL FACS Lyse solution (BD Biosciences, San Jose, U.S.A) for 10 min, in the dark. Cells were then centrifuged at 300 x g for 5 min, washed in 2 mL cell wash, and re-centrifuged at 300 x g for 3 min.
Forward and Side Scatter Optimization
Forward scatter (FSC) amplification (amp) gain and CD45 were adjusted to position the three main leukocyte subpopulations; lymphocytes, monocytes and granulocytes on the linear scale. FSC threshold was adjusted to minimize debris appearing on the scale. FSC and CD45 were optimized for all samples under all conditions. Lymphocyte and monocyte populations were gated using a conjugated-CD45 vs. SSC plot. 1.0 x 106 cells were stained with CD45-PerCP or CD45-PE-CyTM5 to facilitate gating the main types of leukocytes (granulocytes, monocytes and lymphocytes), and to gate out debris. Data acquisition and analysis were completed using the a BD FACS CantoTM II software (CA, USA). The 20,000 events were acquired for each sample.
Expression of surface antigens on monocyte subsets:
The Procedure for analysis of surface antigens used 3 BD Falcon FACS tubes. Tube 1 was the surface negative tube and tube 2 included all antibodies and tube 3 was used as an internal control. Whole blood (100μl) was added to each 3 BD Falcon tubes, then CD 45 was added to all tubes. Next, CD14 and CD16 were added to tube 2 and tube 3. The CD202 was added exclusively to tube 2. All tubes were incubated for 15 minutes in the dark. Then 2ml of BD FACS lysing solution was added to each tube, then incubated for 10 minutes. Then all tubes were washed 2 times with Cell wash PBS. The last step involved adding 500uL to each tube and then it was input using FACS Canto™ II Flow Cytometer.
Statistical Analysis
In this study, Student's T-test was performed to compare the difference in mean SD. GraphPad Prism version 8.00 for Mac Correlation coefficient was made using Person test. Correlation is significant at the p value < 0.05 level (2-tailed). The analysis and graphs were done by, GraphPad Software, La Jolla California USA, www.graphpad.com”.