A Laboratory Approach to Standardize Luminoaggregometry for Sample Shipment

Introduction: Platelet function testing, like the luminoaggregometry for detecting granule secretion disorders and storage pool diseases, usually requires fresh blood samples.

Because of lacking specialized laboratories connected to treating physicians in appropriate time the diagnosis of inherited platelet disorders is challenging. In this pilot study, we validated 48-hour transport of anticoagulated blood for luminoaggregometry.

Methods: Citrated blood of healthy donors was split into to two different tubes. The first one was analysed within 4h after drawing and the second was transported via laboratory transport by car to a collaborating center located 150 km away from our department and returned 36h later to our laboratory. The later sample was analysed 48h after drawing. Luminoaggregometry was performed using platelet rich plasma (PRP) and calibration was performed using an ATP standard. ATP levels were measured after induction with Thrombin Receptor Activating Peptides (TRAPs), collagen and calcium ionophore A23187, respectively.

Results: We shipped blood samples of 30 healthy donors. On day 0 a sufficient aggregation response was observed to TRAP (mean of aggregation: 93%, range 57%-100%) and to collagen (mean of aggregation: 89%, range: 76%-100%)). ATP release was within normal range for 29 samples (mean of release: 2.8nm, range 0.51-7.40nm) after stimulation with TRAP and with collagen (mean of release: 2.1nm, range 0.8-2.0nm). ATP content in the platelets of the healthy donors was also within normal range (Ionophore mean of release: 2.4nm, range 0.97-3.93nm). After two days of shipment we saw reduction in aggregation response to all agonists (TRAP mean of aggregation 67.1%, range 8%-97%, 26/29 samples in normal range; collagen mean of aggregation75%, range 45%-100%; 25/30 samples in normal range). After shipment ATP release decreased non-significant to 1.1nm (0.0-3.18nm, p=0.15) and 1.2nm (0.25-3.73, p=0.11) after stimulation with TRAP and collagen, respectively. Interestingly, 23/30 and 26/30 samples showed results within the normal range for ATP release after shipment when platelets were activated with TRAP and collagen, respectively. ATP content in platelets was slightly reduced after shipment (mean of release: 1.33nm, range 0.25-3.73nm, p=0.12, and 27/30 had concentrations within a normal range after two days of shipment.

Conclusions: Shipment of platelets for luminoaggregometry induces functional impairment in less than 15% of samples. Testing of ATP release in 48h-stored platelets may be useful to rule out ATP release defect or ATP storage defects in platelets. Pathological values should be repeated with fresh blood samples.