Advia 120 hematology system

The volume of the BMA as well as concentration of nucleated cells and platelet retrieved from each bone marrow site was recorded. Nucleated cells and platelet numbers were obtained using the ADVIA® 120 Hematology System (Siemens, Malvern, PA). A subset of cells from BMA and cBMA was seeded onto plastic culture dishes in Dulbecco's Minimum Essential Media (DMEM) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Saint Louis, MO), 2 mM L-glutamine, and 1% penicillin/streptomycin in a humidified incubator (5% CO₂, 37°C). At subconfluency, cells were enzymatically detached with 0.25% trypsin–EDTA and characterized at passage 1 using the following assays: colony-forming unit fibroblast (CFU-F), multidifferentiation capacity, and flow cytometry.

In MARTHA, we used the Agkistrodon contortrix venom (ACV) test as a quantitative biomarker of the protein C pathway. The ACV test was expressed as a normalized ACV value (ACVn) as described in [30] (link). The ACVn ratio was available in 260 MARTHA patients with DNA methylation measurements. A complete blood count, including white blood cell types (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), was determined by ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Deerfield, IL).
In F5L-families, activated protein C resistance (APCR) levels were determined in 208 individuals using the APC-aPTT assay. The results of the test are expressed as the APC-sensitivity ratio, which is the quotient of the activated partial thromboplastin time (aPTT) of the plasma sample with and without exogenous APC [38] (link).

Blood samples
were collected immediately after euthanasia via cardiocentesis into
microtubes containing either EDTA for the hematology analysis or a
serum separator for the blood chemistry analysis. Within 4 h of sample
collection, complete blood counts were analyzed with the ADVIA 120
Hematology System (Siemens). Blood chemistry, including serum levels
of albumin, alkaline phosphatase, alanine transaminase, aspartate
transaminase, blood urea nitrogen, creatinine, globulin, and total
protein, was analyzed using the COBAS INTEGRA 400 Plus system (Roche).

Whole blood was collected either by cardiac puncture or retro-orbital sinus puncture. We mixed whole blood with EDTA (final concentration 5mM) and analyzed complete blood counts using an Advia 120 Hematology system (Siemens, Munich, Germany).
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by standard methods. Bone marrow was obtained by flushing from femur or tibia, and cells were counted using Cellometer^® Auto 2000 (Nexcelom Bioscience, Lawrence, MA, USA) at 1:1 dilution in ViaStain^TM AOPI staining solution (Nexcelom Bioscience). Red blood cells were lysed using red blood cell lysis buffer. Cells were stained with antibody mixes at concentration 1:100 for each surface marker antibody in Hank’s buffered saline solution with 2% fetal bovine serum and 1% HEPES buffer. For Foxp3 staining, cells were stained with surface marker antibodies, fixed using Fixation/Permeabilization (Invitrogen, Carlsbad, CA, USA) diluted in eBioscience^TM Fixation/Perm Diluent (Invitrogen), and stained with Foxp3 antibody at 1:100 concentration in 1× permeabilization buffer (Invitrogen). Multicolor flow cytometry was performed on LRSFortessa cell analyzer (BD Biosciences, San Jose, CA, USA) or LSRII cell analyzer (BD Biosciences), and flow data were analyzed using FlowJo (FlowJo, LLC, Ashland, OR, USA).