3. Study Limitations Related to Platelet Serotonin Platelet-rich plasma Expected fixed platelet count Unstable ACS environment Potential confounders Off-site laboratory processing→ delayed time to processing

4. Platelet Serotonin Laboratory Protocol 1.Remove 400 uL of plasma 2.Add to 15 mL tube for platelet purification 3.Add 1.5 mL physiological saline to platelet- rich plasma (PRP) Centrifuge 10 minutes at 200 RCF(xg) at ambient temperature Centrifuge10 minutes at 4,500 RCF(xg) at 4 o C & discard supernatant 1.Add to 300 uL distilled water 2.Vortex pellet until the platelets are mixed & transfer 300 uL to 1 aliquot Transfer to -80 o C freezer for storage ELISA Immuno Biological Laboratories (2009). Serotonin ELISA IB89546. [Laboratory instructions]. IBL-America 4 mL EDTA tube

5. Storage temperature Duration of storage Freeze-thaw cycles Storage temperature Duration of storage Freeze-thaw cycles Specimen Storage Specimen Storage Instrument calibration & controls Time between collection & centrifugation Vortex technique Centrifugation Type Calibrated Temperature Speed Duration Automated decapping Specimen separated into aliquots Aliquot labeling Instrument calibration & controls Time between collection & centrifugation Vortex technique Centrifugation Type Calibrated Temperature Speed Duration Automated decapping Specimen separated into aliquots Aliquot labeling Processing In Vivo Haemolysis due to patient condition Proper labeling Mode of collection Needle gauge Position of arm Location of venipuncture Antiseptic used Tourniquet time Syringe transfer Specimen tube Type Under/over filled Order of draw Under/over mixing In Vivo Haemolysis due to patient condition Proper labeling Mode of collection Needle gauge Position of arm Location of venipuncture Antiseptic used Tourniquet time Syringe transfer Specimen tube Type Under/over filled Order of draw Under/over mixing Phlebotomy Origin of specimen Vertical or horizontal transportation of tube Pneumatic tube transport Courier transport Transportation duration Centrifugation prior to transfer Transfer temperature Origin of specimen Vertical or horizontal transportation of tube Pneumatic tube transport Courier transport Transportation duration Centrifugation prior to transfer Transfer temperature Specimen Transport Specimen Transport Factors Affecting Biological Specimens