Within the last few years, the platelet function analyzer (PFA-100™) has become increasingly popular in assessing congenital and acquired platelet dysfunction . In this assay, platelet function at high shear condition is evaluated and the closure time (CT) is determined . The PFA-100 ™ uses two types of cartridges where a collagen membrane is coated with either epinephrine (CEPI) or ADP (CADP). Aspirin (ASA) prolongs the CEPI CT, whereas the CADP CT remains normal; thus, the PFA-100™ can differentiate ASA effects from other platelet defects . Whole blood platelet aggregation (WBA) is another method used to assess platelet function and is based on the impedance technique . Both PFA-100™ and WBA use citrated whole blood samples, which should be kept at room temperature and tested within 3–4 h after collection. Blood sample collection and transportation should be performed in a way that avoids platelet activation. Excessive shaking or agitation of the sample can lead to spurious results, due to platelet activation/exhaustion and the presence of air bubbles in the sample. In many hospitals, the requirement of delivering blood specimens to the core laboratory as quickly as possible and of improving laboratory turnaround times resulted in the installation of the pneumatic tube system (PTS) . With the exception of samples for blood gas analysis and evaluation of post-transfusion reactions [4,5], PTS was shown to be a suitable method for blood specimen transportation .
Our goal was to study the effects of PTS transport on PFA-100™ CT and WBA. We studied 27 healthy volunteers (17 males and 10 females) with a mean age of 36 years, who gave informed consent according to an established, Institutional Review Board (IRB)-approved protocol. Seventeen subjects had no history of ingestion of any antiplatelet or anti-inflammatory drug for 14 days prior to the study (non-ASA group). Ten persons took ASA at a dose of 81 mg for 7 days (ASA group). Blood was collected from the antecubital vein into four tubes (3.0 mL greiner bio-one VACUETTE North America, Monroe, NC, USA) containing 300 µL of 3.2% sodium citrate. For the measurement of complete blood count 4 mL of blood were collected in 7.2-mg K2 EDTA Vacutainer® (Becton Dickinson, Franklin Lakes, NJ, USA). Two of four tubes from each collection were packed into two plastic bubble bags and placed in a pneumatic tube container. The specimens were sent from Parkland Hospital's Central Laboratory via PTS to the adjacent Children's Medical Center of Dallas and then were immediately sent back to the Central Laboratory. The total transit time was approximately 15 min, and the distance traveled was approximately 244 m.
In the PFA-100™ study, CTs of CEPI and CADP cartridges were determined using the tubed samples (TS) and non-tubed samples (NTS). TS were again tested after resting at room temperature for 1 h (1 h post-transport sample, 1H-PTS). WBA was performed on the NTS, TS and 1H-PTS in the ASA group and on NTS and TS in the non-ASA group in a Dual Channel Whole Blood Lumiaggregometer (Model 560 VS; Chrono-log Corp., Haverton, PA, USA). Agonists used were ADP (10 µm), collagen (2 mg mL−1) and arachidonic acid (AA; 0.5 mm) (Chrono-log Corp.) as described previously . The ATP secretion during aggregation was assessed by luciferin- luciferase reaction (Luciferin-Luciferase Reagent; Chrono-log Corp.). Statistical analysis was performed using Wilcoxon signed rank test (Stat View Software, SAS, Cary, NC, USA). Study results are given in Table 1.
|Non-ASA||128 ± 53||79 ± 18||129 ± 33||84 ± 18||131 ± 33||85 ± 22||27 ± 6||38 ± 13||12 ± 2||24 ± 4||39 ± 7||10 ± 5||Not done||Not done||Not done|
|ASA||179 ± 89||83 ± 24||201 ± 101||110 ± 39*||191 ± 107||117 ± 42**||2 ± 5||22 ± 7||8 ± 4||2 ± 6||18 ± 8***||7 ± 4||2 ± 5||14 ± 7****||7 ± 3|
In the non-ASA group there was no significant difference between PFA-100™ CTs of (i) NTS and TS and (ii) TS and 1H-PTS. In the ASA group, 9/10 persons had a significant prolongation of CTs with the CADP cartridge in TS and 1H-PTS compared with NTS (P = 0.03; P = 0.01). There was no significant difference in CTs of CEPI cartridges in NTS, TS and 1H-PTS. This is a noteworthy finding, since ASA causes prolongation of CEPI but not that of CADP-CT, therefore making it difficult to interpret the ASA effect if the samples are tubed.
In the non-ASA group, there was no significant difference in aggregation and ATP secretion with AA, collagen and ADP between NTS and TS. In the ASA group, there was a significant decrease in collagen-induced platelet aggregation in 1H-PTS when compared with NTS and TS (P = 0.005; P = 0.01). Although there was decreased collagen-induced aggregation in TS compared with NTS, it did not reach statistical significance (P = 0.07). This indicates that there is exhaustion of platelets during PTS transport, which do not recover even after 1 h of rest. There was no significant difference in ADP-induced platelet aggregation, nor was there a statistical difference in the secretion of ATP with collagen. AA-induced platelet aggregation and ATP secretion in the ASA group was consistent with the ASA effect in all three groups.
Bell et al. and Goldsmith et al. highlighted the role of red blood cells (RBC) in WBA at high shear stress [8–10], observing significantly increased platelet aggregation in WBA compared with platelet-rich plasma at the same shear rate and ADP concentration, which these authors attributed to the mechanical effect of RBCs. They also showed ADP released from RBCs to be a contributing factor . Thus, the release of ADP from hemolyzed or mechanically agitated RBCs during PTS transport may influence platelet function assays. Greendyke et al. reported that hemolysis in samples transported by PTS were rendered unsuitable for assessment of transfusion reactions . In addition, Pragay et al. found significant changes in the activity and level of erythrocyte membrane damage indicators (serum lactate dehydrogenase, acid phosphatase and potassium) in partially filled samples that traveled via PTS . Moreover, Collinson et al. reported that air bubbles found in tubed specimens caused discrepancies in blood gas analysis, affecting pO2 values . This supports our observations, since in our study all tubes contained some air bubbles immediately after being transported through the PTS. In the PFA-100™ system air bubbles may cause flow obstruction and termination of the test. In WBA study, air bubbles surrounding electrodes can create a mechanical interference for platelets.
In conclusion, transportation of blood samples for platelet function studies via tube systems may affect the results of patients who are taking ASA (and possibly other antiplatelet drugs) and may also affect results in patients with primary platelet dysfunction. Thus, hand delivery of whole blood samples to the laboratory is preferable to PTS and highly recommended to obtain reliable platelet assay results.