Morgane Gossez

Francoise Poitevin-Later

Julie Demaret

Laurent Jallades

Fabienne Venet

Christophe Malcus

Guillaume Monneret
email: guillaume.monneret@chu-lyon.fr

Effect of Pneumatic Tube Transport on T Lymphocyte Subsets Analysis

Keywords: certification, flow cytometry, pneumatic tube transport, T lymphocytes

Background: Pneumatic tube system (PTS) for transportation of blood specimens may present with advantages for hospital organization as it provides faster tube transfer from medical wards to routine labs. These characteristics are expected to result in faster sample processing and decreased turnaround time, therefore benefiting the patient particularly in emergency units. However, PTS could affect sample quality and therefore laboratory results. Within the context of routine lab certification, effects of PTS on routine cellular immunology analyses, especially on determination of T lymphocyte subpopulations, need to be evaluated.

Methods: Paired EDTA blood samples were collected from 30 healthy donors. For each pair, one sample was hand-delivered by a courier while the other was transported through a PTS of 2.4 km long (1.6 miles) with two 90-degree turns and one-U turn generating a speed of 5 m/s with a maximal acceleration of 2 g-force. The percentages of CD31 cells, CD41 and CD81 T-cells and their absolute counts were assessed by flow cytometry.

Results: For every parameter, results measured by flow cytometry were not significantly different after transport by PTS or hand-delivery. Results comparison revealed an excellent linear correlation between both delivery methods (R ranged from 0.969 to 0.982; P < 0.01). Bland-Altman plots also showed good agreement, indicating that results were not influenced by the transport method.

Conclusions: Our results suggest that, pending validation using clinical samples, PTS does not present with pre-analytical drawbacks and is thus a reliable system for blood samples transportation when performing T cell subset phenotyping. V

INTRODUCTION

Within the context of hospital resources optimization, there is an increasing development of centralized highly automated and computerized analytical platforms along with implementation of sample transport systems such as pneumatic tube systems (PTS) connecting laboratories and hospital wards. PTS delivery allows for a faster and safer transport method than manual transport by couriers, leading to faster sample processing and decreased turnaround times, which potentially benefits the patient particularly in emergency or critical care units [START_REF] Al-Riyami | The pneumatic tube system does not affect complete blood count results: A validation study at a tertiary care hospital[END_REF][START_REF] Keshgegian | Evaluation of a soft-handling computerized pneumatic tube specimen delivery system. Effects on analytical results and turnaround time[END_REF][START_REF] Fernandes | Pneumatic tube delivery system for blood samples reduces turnaround times without affecting sample quality[END_REF]. In addition, such transport system could be advantageous on situations where rapid transport of samples is of vital importance to reduce decay of lymphocytes such as for cerebrospinal fluid samples. However, this transport method may affect the quality of the samples (pre-analytical step), and thus may have an impact on laboratory results. This mostly depends on PTS technical characteristics, such as length and strong acceleration/deceleration processes [START_REF] Evliyaoglu | Effect of pneumatic tube delivery system rate and distance on hemolysis of blood specimens[END_REF][START_REF] Plebani | Pneumatic tube delivery systems for patient samples: Evidence of quality and quality of evidence[END_REF].

Within the context of laboratory quality management through ISO 15189 (certification process), a well-conducted evaluation of pre-analytical steps (including sample transportation) is mandatory. Some studies have been undertaken about PTS impact on hematology and biochemistry parameters. However to our knowledge, none was designed to assess a putative effect on T cell subsets determination measured by flow cytometry (especially on absolute counting). The purpose of this study was thus to evaluate the effect of a 2.4 km (1.6 miles) long PTS transport (including several curves and acceleration/deceleration steps) on routine immunology parameters. We here focused on T lymphocyte subset analysis, both as percentages and absolute lymphocyte counts.

MATERIAL AND METHODS

Specimens and Transportation

Paired EDTA blood samples were collected from 30 healthy donors (20-61 years old and 15 males and 15 females) in our Academic hospital (900 beds). Paired samples were collected into two vacutainers K 3 EDTA tubes (BD VacutainerV R , Plymouth, UK) from the same venipuncture. Each volunteer provided an informed consent for being enrolled in this study. For each pair, one sample was hand-delivered to the immunology laboratory by a courier (control) while the other was transported through a PTS (H € ORTIG rohrpost, Bayreuth, Germany). PTS length was 2.4 km with two 90-degree turns and one-U turn generating a speed of 5 m/s (i.e., 18 km/h) with a maximal acceleration of 2 g-force (i.e., 19.6 m per second squared). The deceleration is then widespread along the pneumatic length. Sample transport mean time for PTS was 8 minutes and 45 minutes for controls. Paired samples were then labelled independently (PTS vs. CO).

Sample Analysis

Samples were processed simultaneously by the same operator, <6 h after blood collection, and analyzed on the same flow cytometer. Percentages of CD31 cells, CD41 and CD81 T-cells and relative absolute counts were provided by a single-platform bead-based flow cytometry technique (FC500 analyzer, Beckman Coulter, Hialeah, FL). The flow cytometer was daily controlled by running Flow Check Fluorospheres and Immunotrol stabilized blood Cells (BeckmanCoulter). Cytostat Tetrachrom CD45/4/8/3 (Beckman Coulter) was used according to the manufacturer's recommendations. The red cells were lysed on the automated TQ Prep system (Beckman-Coulter). Absolute count was performed using Flow-Count Fluorosphere according to manufacturer's recommendation (Beckman-Coulter). Range of the inhouse coefficients of variation using Immunotrol stabilized blood (BeckmanCoulter) as from 1.04 to 5.12% depending on lymphocyte subsets. The results are expressed as cell percentages among total lymphocytes and as numbers of cells per microliter.

Statistical Analysis

Every variable was normally distributed based on Kolmogorov-Smirnov normality test. Thus, for each parame-ter studied, mean and Standard Deviations (SD) were calculated. Comparisons of results obtained after the 2 transport procedures (PTS vs. controls) were performed using student test. Results agreement was assessed by linear regression analysis and Bland-Altman statistical (6) methods as recommended by the International Federation of Clinical Chemistry and Laboratory Medicine. Pearson correlation coefficient (R) and coefficient of determination (R 2 ) were calculated, and the linearity of the correlation was tested using a student test. Similarly, the slope and the intercept of each linear regression analysis were compared respectively to 1 and 0 by using student tests. Statistical analyses were performed using SPSS 19.0 software (Chicago, IL). Statistical significance was defined by a P values of < 0.05.

RESULTS

As shown in Table 1, mean values of every parameter were not significantly different depending on transportation method. This is graphically illustrated in Figure 1A for CD41 T lymphocytes measurement. Similar patterns were observed for CD31 and CD81 T cells (data not shown). In addition, we observed an excellent linear correlation between both delivery methods (Fig. 1B for CD41 T cells), with correlation coefficients ranging from 0.969 to 0.982 (P < 0.01) and slopes not different from 1. Bland-Altman plots showed that differences in measurements were within the 95% confidence interval of the mean difference, indicating that results were not influenced by the transportation method used (Fig. 1C for CD41 T cells). Nevertheless, means of the differences (represented by the dashed line in Fig. 1C) were systematically lower than zero for every studied parameter indicating a very small but systematic bias. This is in accordance with the observation that intercepts values that were slightly different from 0 (Table 1). That given, as these biases are much lower than the CV obtained for corresponding parameters during method validation, their impact on biological results may probably be negligible (ISO 5275-6)

DISCUSSION

These past few years, within the context of laboratory certification, several studies have been conducted on the influence on biological results quality of sample transportation by PTS.

Indeed, validation of the effects of sample transportation method on biological results generated in routine hospital labs is mandatory with the context of routine lab certification process (ISO 15189).

Moreover, from a clinical perspective, poorly reproducible results may lead to repeated phlebotomies and increased delays in clinical decision-making [START_REF] Kara | Hemolysis associated with pneumatic tube system transport for blood samples[END_REF].

However, results from related studies have been contradictory. Indeed, some authors have reported that samples transported by PTS had a greater frequency of hemolysis than those transported manually leading to abnormalities in potassium and creatinine measurements as well as in parameters of blood gas analysis, white blood cell (WBC) count, WBC differential count, and analysis of platelet function [START_REF] Plebani | Pneumatic tube delivery systems for patient samples: Evidence of quality and quality of evidence[END_REF][START_REF] Kara | Hemolysis associated with pneumatic tube system transport for blood samples[END_REF][START_REF] Thalen | Pneumatic tube transport affects platelet function measured by multiplate electrode aggregometry[END_REF]. In contrast, some authors did not observe any statistical or clinical difference for routine hematology and hemostasis tests between courier and PTS transports [START_REF] Al-Riyami | The pneumatic tube system does not affect complete blood count results: A validation study at a tertiary care hospital[END_REF][START_REF] Keshgegian | Evaluation of a soft-handling computerized pneumatic tube specimen delivery system. Effects on analytical results and turnaround time[END_REF][START_REF] Evliyaoglu | Effect of pneumatic tube delivery system rate and distance on hemolysis of blood specimens[END_REF][START_REF] Plebani | Pneumatic tube delivery systems for patient samples: Evidence of quality and quality of evidence[END_REF][START_REF] Lima-Oliveira | Management of preanalytical phase for routine hematological testing: Is the pneumatic tube system a source of laboratory variability or an important facility tool[END_REF].

These discrepant observations may be due to different local PTS installations. By nature, all PTS are different (in terms of length, curves, speed) as they are strictly dependent on local hospital architectures. Therefore, inhouse/local validation of this method of transportation for every parameter studied in specific routine labs is mandatory. We therefore performed such evaluation for cellular immunology parameters evaluated in our hospital laboratory. Regarding T lymphocyte subsets measurement by flow cytometry, the present study did not show any significant difference between results obtained from both sample delivery methods. We rather observed excellent correlations and agreements. We only observed very small biases in intercepts that however remained largely lower than inherent variability of corresponding techniques. In addition, as results obtained after PTS transportation were slightly higher than those obtained with manual transport, it seems unlikely that PTS may damage lymphocytes.

The strength of our results is that they have been obtained after a quite vigorous transportation: very long PTS (2.4 kms, 1.6 miles) including two 90-degree curves, one U-turn and several acceleration/deceleration phases including a maximal acceleration speed of 2 gforce (i.e., 19.6 meters per second squared). Indeed, in similar studies, high frequency of hemolyzed samples have been reported within such transportation system, potentially impacting laboratory results [START_REF] Lima-Oliveira | Management of preanalytical phase for routine hematological testing: Is the pneumatic tube system a source of laboratory variability or an important facility tool[END_REF][START_REF] Pragay | Evaluation of an improved pneumatic-tube system suitable for transportation of blood specimens[END_REF]. However, as mentioned earlier, our results cannot systematically be extended to every pneumatic system as they do not present with similar specifications.

Our study has some limitations. In particular, our cohort is only composed of healthy volunteers and we cannot exclude that under some particular clinical circumstances results might be different. Therefore a next step will be to confirm our results with clinical samples. In addition as an extended lymphocyte phenotyping has not been performed, we cannot exclude that some minor lymphocyte subpopulations could be affected by PTS transportation. Finally, as different cell types may have different vulnerabilities to PTS transportation, the effect of such transport system should be specifically evaluated on specific subpopulations ideally in combination with functional tests.

CONCLUSION

In this study, we did not observe any difference after PT or CO transportation for routine cellular immunology analysis on healthy volunteers' blood samples. To our knowledge, these results are the first reporting on PTS impact on T-cell subset analyses (especially after a long transportation with several bifurcations and speed shifts) and thus they will likely be helpful within certification process especially regarding CD41 lymphocyte absolute count measurement. Upon further validation in clinical samples, this transportation system appears thus suitable to cellular immunology monitoring in routine lab conditions.

LITERATURE CITED

FIG. 1 .

 1 FIG. 1. Comparison of CD41 T-lymphocyte count (number of cells/mL) in healthy volunteers after transport of blood samples by PTS and by courier (n 5 30). (A) Absolute values of CD41 T cells in samples transported by both methods. No significant difference shown using Student test (P > 0.05). (B) Correlation of CD41 cells absolute count after transportation by courier vs.PTS transportation. (C) Bland-Altman plot analysis of results obtained from paired samples. Values on the x-axis indicate the average of the results from both transport methods for each pair of samples, whereas values of the y-axis represent the differences in the results. Horizontal lines represent mean (-) and 95% limits of agreement (-) of CD41 T cell absolute count differences from both methods of transportation. PTS, Pneumatic tube system; CO, courier; NS, non-significant.

Table 1

 1 Comparison of T-Lymphocyte Subset Results After Blood Samples Transportation By PTS or By Courier

		Transportation	Mean 6 SD	Pearson correlation coefficient R	Slope	Intercept	P value a
	CD31 Ly (%) b	CO	74 6 6	0.982	0.97	1.67	0.125
	CD31 Ly (cells/mL) c	PTS CO	73 6 6 1241 6 294	0.969	0.99	23.53	0.232
	CD41 Ly (%) b	PTS CO	1257 6 302 43 6 6	0.979	0.94	2.39	0.213
	CD41 Ly (cells/mL) c	PTS CO	42 6 6 716 6 179	0.969	1.03	16.06	0.248
	CD81 Ly (%) b	PTS CO	726 6 191 26 6 5	0.967	0.98	0.48	0.887
	CD81 Ly (cells/mL) c	PTS CO	26 6 5 446 6 148	0.979	1.02	0.60	0.140
		PTS	455 6 155
	Results from 30 healthy donors, SD: standard deviation; PTS: Pneumatic tube system; CO: courier, Ly: Lymphocytes. a P-value (PTS vs. CO) from a paired Student test.

b

Percentages among total lymphocytes. c Numbers of cells per microliter.

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