Pneumatic tube system transport does not alter platelet function in optical and whole blood aggregometry, prothrombin time, activated partial thromboplastin time, platelet count and fibrinogen in patients on anti-platelet drug therapy

Introduction The aim of this study was to assess pneumatic tube system (PTS) alteration on platelet function by the light transmission aggregometry (LTA) and whole blood aggregometry (WBA) method, and on the results of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen. Materials and methods Venous blood was collected into six 4.5 mL VACUETTE® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) from 49 intensive care unit (ICU) patients on dual anti-platelet therapy and immediately hand carried to the central laboratory. Blood samples were divided into 2 Groups: Group 1 samples (N = 49) underwent PTS (4 m/s) transport from the central laboratory to the distant laboratory and back to the central laboratory, whereas Group 2 samples (N = 49) were excluded from PTS forces. In both groups, LTA and WBA stimulated with collagen, adenosine-5’-diphosphate (ADP), arachidonic acid (AA) and thrombin-receptor-activated-peptide 6 (TRAP-6) as well as platelet count, PT, APTT, and fibrinogen were performed. Results No statistically significant differences were observed between blood samples with (Group 1) and without (Group 2) PTS transport (P values from 0.064 – 0.968). The AA-induced LTA (bias: 68.57%) exceeded the bias acceptance limit of ≤ 25%. Conclusions Blood sample transportation with computer controlled PTS in our hospital had no statistically significant effects on platelet aggregation determined in patients with anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced.


Introduction
Platelets play a key role in primary hemostasis. The principal function is stopping hemorrhage and preventing blood loss, when vessel walls are injured (1). Platelets interacting with the exposed matrix adhere, become activated, and as a consequence bind further platelets to form a thrombus limited in size. Specific platelet receptors are involved in each of these phases (2).
Platelet function testing has become essential for identifying patients with platelet dysfunction and for monitoring modern anti-platelet therapy. In hemostasis laboratories, platelet aggregometry has been established one of the most widely used platelet function testing procedures (3). Light transmission aggregometry (LTA), which was developed in 1962 by Born and O'Brien, and imped- Enko D. et al. Transport effects on platelet function ance whole blood aggregometry (WBA) are the two main types of methodologies available (3)(4)(5)(6).
Since platelets are sensitive to artificial manipulation, preanalytical variables may influence hemostasis testing in laboratories. The tourniquet application time is reported biasing platelet function testing by multiple electrode aggregometry (MEA) (Multiplate ® ) (7). Moreover, in clinical routine laboratories different vacuum tubes may represent a relevant source of variability on the determination of the mean platelet volume and platelet distribution width (8). Recently, one study concluded that physical exercise in cold water also represents a stress factor, which may cause increased platelet counts (9). Preanalytical variables are often outside the control of hemostasis laboratories and the leading causes of diagnostic error (10). Several crucial steps in the preanalytical phase, such as correct blood collection, blood sample handling, transport and storage of specimens are essential parts to get valid and timely laboratory test results (11).
Over the last years, in many hospitals pneumatic tube systems (PTS) have been established for blood sample transport between departments and outpatient clinics. PTS is considered an efficient and cost effective transport solution specially designed to handle automated blood specimen transport in routine and emergency settings. Nev-ertheless, blood samples transported by PTS are often subjected to high speed (up to 7.6 m/s) accompanied by rapid acceleration and deceleration causing shear stress (12). Changes in air pressure, vibrations and shaking of blood samples are considered to affect various laboratory parameters including coagulation assays (13).  Table 1.
The aim of the present study was to investigate the effect of PTS on the results of LTA, WBA, platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen.

Blood sampling
Blood sampling was done in the morning without obligatory fasting state. From all 49 study participants venous blood was collected into six 4.5 mL VACUETTE ® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria). Immediately after the venipuncture, all vacuum tubes (6 x 49) were gently inverted five times and hand carried by our laboratory personal to the core laboratory of our hospital. In the central laboratory blood samples were divided in Group 1 (3 x 49 tubes) and Group 2 (3 x 49 tubes) without delay. Group

Pneumatic tube system (PTS)
Blood samples of Group 1 (3 x 49 tubes) were transported by a computer controlled PTS (Swisslog Rohrpostsysteme GmbH, Westerstede, Germany) using standard pneumatic tube carriers. One distance was about 500 m with a mean transit time of 2 min. As a result Group 1 samples (3 x 49 tubes) were subjected a twice done acceleration and deceleration during transportation process with PTS, whereas Group 2 samples (3 x 49 tubes) were excluded from PTS transport.
After computer controlled acceleration an average velocity of 4m/s was used. Special receiving stations with controlled deceleration delivered softly the pneumatic tube carrier on special exit conveyor belts.

Statistical analysis
The distribution of data was calculated with the Kolmogorov-Smirnov test. The non-parametric Wilcoxon test was used for comparisons of parameters between Group 1 and Group 2. Not normally distributed data were described in medians (Q1 -Q3). A P-value < 0.05 was considered statistically significant. Bias calculation was performed and compared with available acceptance criteria in accordance with the Milano hierarchy (16). The calculated biases were compared to the acceptance limits based on the Clinical Laboratory Improvement Amendments (CLIA) for analytical quality (17). SPSS Statistics for Windows version 22.0 (IBM SPSS Inc., Chicago, Illinois, USA) was used for statistical analysis.

Results
The results of platelet function testing with LTA and WBA, and the coagulation parameters (i.e., platelet count, PT, APTT, and fibrinogen) between blood samples of Group 1 (N = 49) and Group 2 (N Enko D. et al. Transport effects on platelet function = 49) and the mean bias are shown in Table 2. All parameters investigated in the present study were not normally distributed and are presented in medians (Q1 -Q3). No statistically significant differences were observed between blood samples transported with (Group 1) and without PTS (Group 2) (P values from 0.064 -0.968).

Discussion
To the best of our knowledge, this is the first study reporting data about PTS effects on both, optical aggregometry as well as impedance aggregrometry measurements in patients on anti-platelet drug therapy. The results referred only to patients on dual acetyl-salicylate acid and clopidogrel therapy   In comparison, previously published studies on the subject of PTS effects on platelet aggregation tests were designed with 12 to 58 subjects (18 -23) ( Table 1). Two studies, comprising 58 and 50 individuals, observed significant influence on impedance WBA with a Multiplate ® analyzer (20,23). In both studies, detailed information about speed of sample transport and other characteristics of the PTS, that may strongly affect pre-analytical quality are lacking. Reducing speed is considered to avoid pre-analytical alteration of blood samples (24 (23). Another study on the Multiplate ® system evaluated 50 patients with acetyl-salicylate acid therapy using AA (0.5 mmol/L) and TRAP (32 µmol/L) only (20).

AA-induced
Considering these various study designs performed with different agonists and concentrations, the major limitation of data comparison is the lack of standardization in platelet function testing. Widespread accepted uniform guidelines on how laboratories should perform clinical testing for disorders of platelet function are still not available (26,27). Results of two surveys of the North American Specialized Coagulation Laboratory Association (NASCOLA) demonstrated that agonist concentrations varied widely and that various methods were used to obtain reference intervals for platelet aggregation testing (26). Although LTA is considered to be the "gold standard" for in vitro platelet function testing in both clinical and research laboratories, no evidence-based guidelines for the performance and interpretation of studies with this technique are established yet (28,29). Moreover no proper commercial controls are available for the different platelet function assays to examine the validity of the test results.
In the present study the mean bias of the investigated parameters between blood samples transported with (Group 1) and without PTS (Group 2) ranged from 0.0 to 68.57%. Compared to the available acceptance criteria in accordance with the Milano hierarchy (16,17), analytical performance evaluation studies may be influenced by the measurement quality, the actual test method used, and the investigated study population. Therefore standardized platelet function testing procedures with commercially available QC material for LTA and WBA is required in order to improve analytical quality and reproducibility.

Enko D. et al. Transport effects on platelet function
The major limitation of this study is that healthy subjects, who were not on anti-platelet therapy, were not included. We cannot omit a protective effect on acetyl-salicylate acid and clopidogrel during transportation. Nevertheless, the added value of this study is that potential PTS effects were investigated on platelet function testing with the LTA as well as the WBA method.
In conclusion, blood sample transportation with soft motion computer controlled PTS, as installed in our hospital, had no statistically significant effects on platelet aggregation measured with optical and impedance aggregometry in patients with dual anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced at all. Further investigation on healthy subjects without anti-platelet therapy should be performed.