An In Vitro Model for Studying Neutrophil Activation During Cardiopulmonary Bypass by Using a Polymerase Chain Reaction Thermocycler

Abstract

The accurate temperature control of a polymerase chain reaction (PCR) thermocycler was exploited in developing an in vitro model to study neutrophil activation during cardiopulmonary bypass. Neutrophils from 12 volunteers underwent temperature changes in a PCR thermocycler (37°C for 30 minutes, 28°C for 60 minutes, and then 37°C for 90 minutes). Different co-incubates were applied to neutrophils, as follows: Group A: phosphate-buffered saline solution; Group B: platelet activating factor (PAF); Group C: platelet-depleted plasma; Group D: platelet-depleted plasma + PAF; and Group E: platelet-rich plasma. Membrane-bound elastase (MBE) activity was measured every 30 minutes throughout the experiment. MBE acticity decreased significantly after hypothermia, compared with the baseline level (p < 0.001), and it resumed an increase after re-warming. Among all co-incubates, platelet-rich plasma was the most potent pro-inflammatory stimulus to neutrophils. A linear correlation was found between MBE and platelet count in platelet-rich plasma (p = 0.004). A novel in vitro model involving a PCR thermocycler has been proved to be reliable in the study of neutrophil activation during cardiopulmonary bypass. The model could possibly be used as an alternative to animals in the development of new drugs to combat neutrophil damage to tissues and organs during cardiopulmonary bypass in cardiac surgery.

Aggressive re-warming at 38.5 °C following deep hypothermia at 21 °C increases neutrophil membrane bound elastase activity and pro-inflammatory factor release

“…Neutrophils were incubated with either PBS or PRP in each temperature treatment. Thermocycler (Gene Amp ® 2400, Applied Biosystem, USA) for polymerase chain reaction was used for the hypothermia and re-warming treatments according to our previous description (Tang et al 2004 , 2010 ). Aliquots of 50 μL neutrophil suspension either in PBS or mixed with PRP was prepared.…”