A performance evaluation of chemiluminescence enzyme immunoassays on the Sysmex CN‐6500 haemostasis analyser

Abstract Background The Sysmex CN‐6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. Aims To evaluate the analytical performance of the CN‐6500 against the predicate device (Sysmex HISCL‐800) for soluble thrombomodulin (TM), thrombin‐antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI‐C) and plasmin α2 plasmin inhibitor complex (PIC) assays. Methods Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. Results The CN‐6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between‐run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89‐1.03; r2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI‐C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed. Conclusions Our data demonstrate that the performance of the CLEIA assays on the CN‐6500 is comparable to that of a stand‐alone immunoassay analyser.


| INTRODUC TI ON
The Sysmex CN-series (Sysmex Corporation) is a range of highthroughput compact haemostasis analysers. 1 The CN-6500 analyser has an integral immunoassay module which performs chemiluminescence enzyme immunoassays (CLEIA) in addition to coagulation, chromogenic, immunoturbidometric and light transmission platelet aggregometry testing. CLEIA is an immunoassay technique in which the label is a luminescent molecule. It typically offers a wide linear measurement range, with a high signal intensity, and an absence of interference, resulting in high sensitivity and specificity. 2 In common with other automated CLEIA systems, the CS-6500 is much faster than traditional immunoassays.
The CN-6500 currently has four CLEIA assays available; soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin/α2 plasmin inhibitor complex (PIC). The performance of these assays has previously been evaluated on the HISCL-5000 (Sysmex Corporation), 3 a stand-alone fully automated rapid immunochemistry system which utilizes CLEIA technology. The HISCL system uses biotinylated primary antibodies, streptavidin-coated magnetic beads, alkaline phosphatase (ALP) labelled secondary antibodies and an ALP chemiluminescent substrate (CDP-Star®). These assays are reported to have clinical utility in the diagnosis of disseminated intravascular coagulation (DIC) and its subtypes. [3][4][5] They also have potential applications in cancer medicine. 6 The diagnostic criteria for DIC proposed by the Japanese Society for Thrombosis and Haemostasis recognize different subtypes of DIC, which reflect fibrinolytic status. 4 Consequently, the measurement of TAT and PIC is frequently included in the diagnostic workup for DIC in Japanese laboratories. Endogenous soluble TM may be assayed as a marker of endothelial injury, as it is cleaved and released from the vessel wall in many inflammatory conditions. 7 tPAI-C may also be a useful assay in the investigation of DIC, and increased levels are associated with multiorgan failure. 8 More recently, it has been observed that TM, TAT, PIC and tPAI-C may have prognostic value in assessing patients with cancer 6 COVID-19. 9,10 The purpose of this study was to compare the performance of four CLEIA tests on the CN-6500 haemostasis analyser against the same reagents on the predicate device (HISCL-800).

| ME THODS
The analytical performance of the CN-CLEIA system was compared with that of a stand-alone immunoanalyser (Sysmex HISCL-800) for TM, TAT, PIC and tPAI-C. 6,7 The CN-6500 performed the CLEIA method as follows: 10-30μl of sample was incubated with a primary biotinylated antibody in the liquid phase to allow antibody-antigen binding. Streptavidin-coated magnetic beads were added to bind antibodies, and free antigen was removed by washing. Following a brief incubation with an ALP labelled secondary, unbound secondary antibody is removed by washing. Buffer was added to disperse the beads, the ALP chemiluminescent substrate (CDP-Star®) was added and luminescence intensity measured. TAT and tPAI-C were reported in ng/ml, TM was reported in TU/ml (thrombomodulin units), and PIC was reported in µg/ml, as per the manufacturer's instructions. Precision was assessed using Controls L and H (normal and high QC materials) provided by the manufacturer tested 10 times on each of 5 separate days. BioLogic Inc) were also tested. Informed consent was obtained from normal donors (approved by the UCL Research Ethics committee: Project ID Number: 7029/001).
Linearity, lower limit of detection (LLoD) and lower limit of quantitation (LLoQ) were determined by serial dilution of pathological plasma samples in assay diluent across the reportable range. For LLoD and LLoQ determination, each dilution was tested six times.
The LLoD was the lowest concentration showing a statistically significant difference from the assay blank. This was achieved by determining the mean concentration at each dilution and performing a T test relative to six blank measurements. The LLoQ was the lowest concentration which gave a %CV of <10%).
Sample carryover was investigated as previously described. 11 Briefly, aliquots of normal plasma (A1-A3) and plasma containing high levels of the analyte of interest (B1-B3) were tested in the sequence A1, A2, A3, B1, B2, B3 five times, then repeated with the sequence in reverse.

| RE SULTS
Ten replicate tests of high and low QC preparations tested over 5 days gave coefficients of variation of <4.0% for both within-and between-run imprecision ( Table 1)

| D ISCUSS I ON AND CON CLUS I ON S
The CN-6500 is the first routine haemostasis analyser to incorporate a chemiluminescence module. At present, only TM, TAT, PIC and tPAI-C assays are available on the CN-6500 but it is expected that other assays will be added following this successful proof of concept.
At present, twenty immunoassays are available on the HISCL-800 for a wide range of analytes associated with coagulation 5,14 (eg PIVKA), tumour markers, virology and immunology. 15 17 We would recommend establishment of a local RI in at least 120 normal healthy individuals representative of the local population prior to introducing these assays into clinical use.
The correlation between the HISCL-800 and CN-6500 was excellent in a wide range of clinical samples covering the reportable range for all four CLEIA parameters and gave results that were comparable to a previous study. 5 However, the CN-6500 produced t-PAIC values with a significant bias, which indicates a problem with the calibration of this assay. However, agreement within the reference interval was acceptable (Figure 3), so the differences at higher concentrations are unlikely to be of clinical significance. Nevertheless, we have raised concerns regarding possible calibration issues with the manufacturer.
We conclude that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser.

ACK N OWLED G EM ENTS
We are grateful to Dr Steve Kitchen, from the Royal Hallamshire Hospital, for providing samples for this study.

CO N FLI C T S O F I NTE R E S T
This work was supported by an institutional, unrestricted research grant from Sysmex UK. CG, IJM and SJM are consultants for Sysmex Corp.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.