Effect of Pneumatic Tubing System Transport on Platelet Apheresis Units

Abstract

Platelet apheresis units are transfused into patients to mitigate or prevent bleeding. In a hospital, platelet apheresis units are transported from the transfusion service to the healthcare teams via two methods: a pneumatic tubing system (PTS) or ambulatory transport. Whether PTS transport affects the activity and utility of platelet apheresis units is unclear. We quantified the gravitational forces and transport time associated with PTS and ambulatory transport within our hospital. Washed platelets and supernatants were prepared from platelet apheresis units prior to transport as well as following ambulatory or PTS transport. For each group, we compared resting and agonist-induced platelet activity and platelet aggregate formation on collagen or von Willebrand factor (VWF) under shear, platelet VWF-receptor expression and VWF multimer levels. Subjection of platelet apheresis units to rapid acceleration/deceleration forces during PTS transport did not pre-activate platelets or their ability to activate in response to platelet agonists as compared to ambulatory transport. Platelets within platelet apheresis units transported via PTS retained their ability to adhere to surfaces of VWF and collagen under shear, although platelet aggregation on collagen and VWF was diminished as compared to ambulatory transport. VWF multimer levels and platelet GPIb receptor expression was unaffected by PTS transport as compared to ambulatory transport. Subjection of platelet apheresis units to PTS transport did not significantly affect the baseline or agonist-induced levels of platelet activation as compared to ambulatory transport. Our case study suggests that PTS transport may not significantly affect the hemostatic potential of platelets within platelet apheresis units.

Keywords: Hemodynamics; Hemostasis; Platelet apheresis units; Platelet function; Pneumatic tubing system.

Figure 1. A flow chart of an experimental platelet apheresis unit handling procedure

A flow chart describing events on the day of an experiment. Prior to transport, a basal sample was extracted from an apheresis bag containing a platelet apheresis unit. Subsequently, the remainder of the platelet apheresis unit was split into two apheresis bags and transported to the OHSU Trauma and Surgical Intensive Care Unit (ICU) via two parallel methods: pneumatic tubing system (PTS; Swisslog TransLogic, Apeldoorn, The Netherlands) or ambulatory (AMB) transport. All samples were subsequently processed at the research lab. When appropriate washed platelet and supernatants were purified from samples.

Figure 2. Physical parameters of platelet apheresis unit handling

Acceleration/deceleration profiles generated during transport via pneumatic tubing system (A; n = 16), ambulatory transport (B; n = 16) or platelet centrifugation (C; n = 5) as measured using an accelerometer. Representative z-coordinate tracings are shown. Data are reported as mean±SEM.

Figure 3. Effect of transport on platelet activation and aggregation

Washed platelets isolated from platelet apheresis units were collected before (basal) or following transport via pneumatic tubing system transport (PTS) or by human courier, ambulatory transport (AMB). Samples were incubated with indicated agonists for 30 minutes, immunostained and evaluated by fluorescence-activated cell sorting (FACS) cytometry for percent platelet activation (CD41+/CD31+/CD62P+ events; A) or platelet microaggregate formation (high fluorescence intensity CD41+/CD31+ events; B). Mean±SEM, n = 7.

Figure 4. Effect of platelet apheresis unit supernatant on fresh platelet activation and aggregation

Platelet apheresis units were collected before (basal) or following transport via pneumatic tubing system transport (PTS) or by human courier, ambulatory transport (AMB) and pelleted by centrifugation to isolate supernatants. Freshly prepared washed platelets were resuspended in serum supplemented with indicated fraction (percent total volume) of platelet apheresis unit supernatants. As a control, freshly washed platelets were resuspended in serum supplemented with supernatants isolated from cPRP. Fresh platelet activation (A) and microaggregate formation (B) in the presence of indicated levels of platelet supernatants were quantified by FACS; Mean±SEM, n = 4.

Figure 5. Effect of platelet apheresis unit handling on platelet binding to collagen and VWF under shear

Platelet apheresis units were collected before (basal) or following transport via pneumatic tubing system transport (PTS) or by human courier, ambulatory transport (AMB). Platelets content within platelet apheresis units was quantified and final platelet counts were adjusted to 4×10⁵ plts/μL using autologous supernatants. Samples were perfused over surfaces of immobilized collagen, VWF or fibrinogen at a shear rate of 300 s⁻¹ for 10 minutes. Differential interference contrast (DIC) microscopy of platelet adhesion and aggregation representative of three separate experiments (A) and surface area quantification mean±SEM (B) are shown. *, **, ^# and ^## indicate statistically different groups with corresponding p-values of 0.010, 0.013, 0.027 and 0.003, respectively.

Figure 6. Effect of platelet apheresis unit handling on levels of platelet GPIb receptor and VWF forms

Platelet apheresis units were collected before (basal) or following transport via pneumatic tubing system transport (PTS) or by human courier, ambulatory transport (AMB) and were immunostained for surface expression of GPIb and evaluated by FACS. Geometric mean fluorescent intensity (GeoMFI) of GPIb levels were normalized to levels found in freshly prepared cPRP samples. Mean±SEM, n = 4. (A). In select experiments, platelet apheresis units and cPRP samples were pelleted by centrifugation to isolate and test supernatants for VWF multimers by Western blot. Total levels of VWF forms were normalized to vinculin (B; ns = not statistically significant with p = 0.1173, ** p = 0.0331 and *** p = 0.0123; n = 4). Ratios of VWF forms, high molecular weight multimer (HMWM), dimer and mature, were normalized to mature VWF forms (C; n = 4).