Multicentre verification of haematology laboratory blood collection tubes during a global blood collection tube shortage

Verification of blood collection tubes is essential for clinical laboratories. The aim of this study was to assess performance of candidate tubes from four alternative suppliers for routine diagnostic haematology testing during an impending global shortage of blood collection tubes.


| INTRODUCTION
Accurate and reliable laboratory test reporting is crucial for clinical decision-making related to admission and discharge of patients from hospital as well as administration of medications and/or blood products. 1 The quality of the reported test result is directly linked to the quality of the specimen, therefore, unsuitable samples can compromise the accuracy of reported results. 2 Errors may occur in the preanalytical, analytical and post-analytical phases of testing. Technological advances have reduced the errors in the analytical and postanalytical phases. 3 Control of errors within the pre-analytical phase, where the majority of errors occur, is difficult as the pre-analytical phase often involves non-laboratory personnel and also escapes quality control and proficiency testing programmes. 3,4 An important preanalytical consideration is that blood collection tubes are important sources of laboratory variability. 5,6 Thus, verification or validation studies are required prior to the use of new brands of blood collection tubes. 7 Limited studies have been published that assess the differences in the performance of different blood collection tube brands within the haematology context.
To our knowledge, only one study has assessed different brands of dipotassium ethylenediaminetetraacetic acid (K 2 EDTA) blood collection tubes as a source of pre-analytical variability. 8 The full blood count (FBC) and differential white cell count (Diff) parameters of BD A few studies have assessed different brands of sodium citrate blood collection tubes as a source of pre-analytical variability. Adequate filling of citrate tubes to maintain the 9:1 ratio of blood to buffered citrate is pertinent to the accuracy of clotting times. 5,6 Filling accuracy was found to be acceptable for sodium citrate tubes (3.2%) of BD Vacutainer ® , Greiner Bio-One Vacuette ® and Vacutest ® Kima (Vacutest Kima SRL, Arzegrande, Italy), but there was considerable between-lot bias, approaching 13% in the BD Vacutainer ® tubes. 9 A study assessing sodium citrate (2.8%) blood collection tubes by measuring clotting times of recalcified blood and plasma samples using free oscillation rheometry, showed that BD Vacutainer ® and Greiner Bio-One Vacuette ® tubes had shorter clotting times than S-Monovette ® tubes (Sarstedt, Nümbrecht, Germany), which was not due to the anticoagulant solution. 10 Another study, which assessed prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen on the ACL TOP coagulation analyser (Instrumentation Laboratory, Milan, Italy), showed significant differences in PT and aPTT, but not fibrinogen, between sodium citrate (3.2%) blood collection tubes of Venosafe™, Greiner Bio-one Vacuette ® , BD Vacutainer ® , S-Monovette ® and LABOR IMPORT (Shandong Weigao Group Medical Polymer, People's Republic of China). 11 The variation in results for different blood collection tube brands reported in the above studies supports the need blood tube verification in the haematology laboratory.
Early in the COVID-19 pandemic, blood collection tube manufacturers experienced transportation delays, shortage of production inputs and increased cost of raw materials. This led to impending shortages of the widely used BD Vacutainer ® blood collection tubes. 12 The aim of this study was therefore to verify alternative K 2 EDTA and sodium citrate tubes for routine haematology testing, in accordance with published recommendations. 13,14 2 | MATERIALS AND METHODS

| Study design
A multicentre blood collection tube verification study was performed to assess technical and clinical aspects of disposable vacuum blood collection tubes of four manufacturers. The suppliers provided K 2 EDTA and sodium citrate (3.2%) tubes with similar anticoagulant concentrations to the in-use BD Vacutainer ® comparator tubes, as per recommendation. 15 Serum tubes with gel separator, lithium heparin tubes, sodium fluoride tubes and plasma tubes with gel separator were also assessed for each volunteer as part of the broader study.
These findings will be reported separately.
The study was conducted between September 2021 and December 2021 at the National Health Laboratory Service (NHLS) branches of two large academic hospitals in Cape Town, South Africa, namely Groote Schuur Hospital (GSH) and Tygerberg Hospital (TBH) and a regional laboratory in Green Point (GPL). All three laboratories are accredited according to international standards by the South African National Accreditation System (SANAS). This verification was performed in accordance with NHLS standard operating procedures, the Clinical and Laboratory Standards Institute (CLSI) (GP41 for sample collection; EP09c for assessment of bias) and published literature. 16

| Collection of blood samples
The collection of blood samples was performed by NHLS trained phlebotomists, according to the recommendations of the Clinical Laboratory Standard Institute (CLSI). 17 The four candidate tube manufacturers that were assessed are presented in Table 1.
Sodium citrate (3.2%) and K 2 EDTA candidate tubes were tested in parallel with the comparator BD Vacutainer ® sodium citrate (3.2%) and K 2 EDTA tubes. Blood was collected from 25 volunteers for each of the four candidate tube manufacturers at each study site. Blood was collected from the anterior cubital fossae of each arm into the comparator and candidate tubes respectively. A 21-or 23-gauge needle was used, which was connected directly to the blood collection tubes via a BD Vacutainer ® holder. In volunteers with difficult venous access a butterfly needle and/or dorsa of the hands were utilized to acquire samples.
The order in which the comparator and the candidate tubes were drawn was alternated to prevent bias, in keeping with guidelines. 18

| Technical verification
Prior to blood collection, all candidate and comparator tubes were assessed for physical defects of manufacturing. Each comparator and candidate tube pack received a unique number, which was recorded during phlebotomy. During phlebotomy, fitting and difficulty with insertion of blood tubes into the collection device, defective vacuum, external blood contamination and any difficulty with phlebotomy which may affect the Blood films were prepared from K 2 EDTA samples. At GSH, blood films were made and stained by an automated process on the Sysmex XN9000 slide-making/staining module (SP-10) (Sysmex Corporation, Kobe, Japan). At TBH, blood films were made manually and stained on the HemaTek (Paderborn, Nordrhein-Westfalen, Germany) flat-bed stainer within 2 h of blood collection. The Wright-Giemsa stained blood films were assessed for staining quality, specifically assessing for adequate staining of cellular components such as nuclear chromatin and granules and EDTA-related changes.

| Clinical verification
All reagents and controls were stored and handled appropriately as per standard laboratory practice and manufacturer's specifications. The K 2 EDTA tubes of the comparator and candidate tubes were assessed for an automated full blood count (FBC), differential count and reticulocyte count on the Sysmex XN2000 (Sysmex Corporation, Kobe, Japan) at GSH and on the Siemens Advia 2020i (Siemens HealthCare Diagnostics, Erlangen, Germany) at TBH. At GPL, FBC parameters for the EV and LG studies were analysed on the Sysmex XN2000 ( Table 1).
The sodium citrate tubes of the comparator and candidate blood tubes were assessed for an automated prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimers on the ACL TOP 500 CTS (Werfen Group, Barcelona, Spain) at GSH and the Sysmex CS-2100i (Sysmex Corporation, Kobe, Japan) at TBH. Sodium citrate tubes were not analysed at GPL.

| Statistical analysis
Data analysis was performed using Microsoft Excel ® (Microsoft Corp., Redmond, Washington, USA) and EP Evaluator™ (Data Innovations T A B L E 1 Candidate K 2 EDTA and sodium citrate tubes assessed within the multicentre blood collection tube verification study. No major problems were identified during phlebotomy for BD, EV and VT tubes ( Table 2). The LG tubes had more angular sideways movement in the blood collection barrel because of the shorter cap.
Additionally, the LG tubes had a small amount of blood leaking from the tube caps where the rubber had been pierced. The rubber of the KV tube caps was hard, which made it more difficult to pierce than other tubes and created more resistance to tube removal. The force required to insert and remove tubes into and from the blood collection barrel occasionally displaced the needle during phlebotomy.
Under-filling was seen in less than 10% each of the comparator tubes.
Stain quality was acceptable for slides prepared from samples in all candidate tubes and the comparator tubes with adequate staining of cellular components such as nuclear chromatin and granules, and no EDTA-related changes seen. The percentage of errors was less than 5% for all tubes (   (Table 3).
A sub-analysis of Bland-Altman plots of PT and aPTT at GSH and TBH was performed. There was a difference in the range of results obtained for PT and aPTT at each of the centres (x-axis, Figure 1). A similar average %bias was seen at TBH and GSH for PT in the LG tubes (y-axis, Figure 1). However, %bias was negative at TBH in EV and KV tubes and positive in VT tubes, while the opposite was seen in T A B L E 2 Technical aspects of K 2 EDTA and sodium citrate tubes for verification.  LG 68  these tubes at GSH. Similar average %bias was seen at TBH and GSH for aPTT in EV, LG and KV tubes. In the VT tubes, the %bias was more negative at GSH (Figure 1).

| DISCUSSION
Blood collection tubes are an often overlooked cause of variability in the pre-analytic phase of laboratory testing. 21 This study showed that the selected tube brand influences the results obtained in routine haematology testing.
For the clinical verification of K 2 EDTA tubes, the EV, LG and KV tubes had acceptable results in comparison to BD tubes. VT K 2 EDTA tubes did not meet the standard for clinical verification, as the MCV and MCHC had unacceptable bias. Since MCHC is calculated from the measured MCV, the bias in the MCV is likely responsible for the bias in the MCHC. A previous study has also demonstrated differences in MCV between different tubes, though VT tubes were not included in their study. 8 For the clinical verification of sodium citrate tubes, none of the tubes had acceptable results in comparison to BD tubes. There was significant bias seen for PT and PTT, though results were acceptable for fibrinogen. Differences in PT and aPTT across different brands of sodium citrate blood collection tubes was also found in an Italian study performed on the ACL TOP analyser. 11 It should be noted that for the aPTT of all the candidate tubes, the mean %bias was greater than the desirable %bias. However, for LG 50   the LG and VT tubes the 95% CI of the %bias crossed the desirable % bias limit and were therefore considered acceptable in accordance with the guidelines. 16 The degree of %bias is nonetheless of concern and if one of these candidate blood collection tubes were to be used, tube specific verification of reference ranges would be needed.
The reason for performing a multicentre verification study was to ensure that the results obtained for the candidate tubes were  experienced phlebotomists in a controlled environment with healthy volunteers, the difficulty would likely be amplified in pressurised clinical situations. While the angular movement of LG tubes within the BD barrel was not a problem for the phlebotomists in this study, it may lead to difficulties in users with less expertise. Additionally, the LG tubes had external blood contamination from the rubber stopper at the site of piercing which is of concern from a health and safety perspective. It should be noted that a BD collection device was used for all candidate tubes, as the purpose of this verification study was to find an interim tube replacement for an impending BD blood collection tube shortage. Thus we cannot confirm whether these technical concerns are valid if collection devices specific to the candidate tubes were to be used.
The limitations of the study include that only healthy volunteers were included and as such, for some of the parameters the correla-

AUTHOR CONTRIBUTIONS
Erica-Mari Nell partook in data collection and data analysis, and was the primary author responsible for writing the manuscript. Jenique Bailly, Diana Oelofse, and Michael Linström partook in data collection and data analysis and contributed to writing the manuscript. Jessica Opie and Zivanai Cuthbert Chapanduka were involved in oversight of the study and contributed to writing the manuscript. Marizna Korf co-designed the study, partook in data collection, was the primary author performing the data analysis, assisted in critical appraisal of the study and contributed to the manuscript. Helena Vreede co-designed the study, advised with data analysis, assisted in critical appraisal of the study and contributed to writing of the manuscript.