Metrological traceability in flow cytometry? Evaluation of a new volumetric method for lymphocyte subsets

Lymphocyte subset enumeration by flow cytometry is important for the therapeutic monitoring of a range of conditions. However, current bead‐based methodologies do not produce metrologically traceable results. Here we compare an established bead‐based methodology with a volumetric‐based system traceable to an internationally recognised reference method.


| INTRODUCTION
The enumeration of lymphocyte subsets is well-established in the field of flow cytometry and is undertaken in a variety of clinical settings.It is most often associated with CD3 + CD4+ lymphocyte monitoring in the HIV+ patient group, where it is used to guide therapeutic interventions. 1,2However, lymphocyte subset results are also essential in the investigation of primary immunodeficiencies, 3 for the monitoring of immunosuppressive therapies 4 and to observe immune reconstitution post allogeneic haemopoietic stem cell transplantation. 5,68][9] Guidelines 10,11 and publications from external quality assessment (EQA) providers 8,9,12,13 recommend that lymphocyte subsets are undertaken using a single platform approach.For a methodology to be classed as a single platform, all results for a patient sample should be generated using just the flow cytometer, this is achieved using either a bead-based or volumetric approach. 7,14,15Results from the UK National External Quality Assessment Scheme for Leucocyte Immunophenotyping (UK NEQAS LI) Immune Monitoring EQA scheme show that participating laboratories predominantly use beadbased assays over volumetric methods (Personal communication).
Regardless of the methodology and diagnostic instrumentation used; the results of laboratory investigations need to be comparable.
7][18] This can be achieved if the results obtained are metrologically traceable.
Metrological traceability is defined as the "Property of measurement result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty." 19Equivalent results may be obtained by establishing metrological traceability of the numerical values that are assigned to the calibrators to the highest available reference system. 20e reference system may comprise reference measurement procedures, certified reference materials or harmonisation reference protocols. 20trological traceability, equipment calibration and measurement uncertainty constitute part of the requirements for accreditation to ISO 15189:2012, which is an internationally recognised standard for medical laboratories.Laboratory accreditation to this standard is the formal recognition that a laboratory has the technical competence and an established quality management system that meets the requirements to deliver technically valid results. 21,22Clause 5.3.1.4 of ISO 15189:2012 relates to equipment calibration and metrological traceability.It requires the laboratory to have documented procedures for the calibration of equipment where it has a direct or indirect effect on examination results and to record the traceable calibration of the item of equipment and the metrological traceability of the calibration standard. 21,23However, it is recognised within the standard that this may not be possible and alternative evidence for providing confidence in the results may be provided.
In flow cytometry, metrological traceability has not been defined in any area.The closest that flow cytometry has come to a reference material was the first WHO International reference reagent for human CD4 T-cells, NIBSC code: 15/270, although in the package insert it is stated that it is not suitable for use as a calibrator as the evidence from the validation study was not strong enough to support its use as an international standard. 24Therefore, alternative evidence is required for current flow cytometric investigations to be compliant with ISO 15189:2012.According to the ISO 15189:2012 standards, such evidence may include "the use of certified reference materials, examination or calibration by another procedure, mutual consent standards or methods which are clearly established, specified, characterised, and mutually agreed upon by all parties concerned." 21Within flow cytometry the use of best practice guidelines for a particular method, combined with process controls for internal quality control, and participation in an accredited EQA scheme are examples of alternative evidence that provide confidence in the results issued. 8,10,11recently launched clinical flow cytometer (XF-1600, Sysmex Corporation, Kobe, Japan) has the capability to obtain absolute lymphocyte subsets using a volumetric-based technique, which incorporates a calibrated syringe-based measurement of the white blood cell (WBC) count on the flow cytometer.The assigned value of the calibrant used is traceable to an internationally recognised reference method for WBC, according to the recommendations of the International Council for Standardization in Haematology (ICSH). 25This measured WBC count is then used in conjunction with lymphocyte and subset percentages to obtain absolute counts, producing a metrologically traceable method for lymphocyte subsets.
This study compared lymphocyte subsets obtained with the Sysmex XF-1600 method with an established CE-IVD labelled 6-colour TBNK assay.The aim was to assess whether the results obtained from this first-stage evaluation were comparable to a widely accepted and established bead-based technique to establish a proofof-concept as to whether metrological traceability in flow cytometry can be achieved.For laboratory implementation and accreditation, further validation would be required, but this was outside the scope of this work.

| Sample preparation
The samples used in this study were from patients attending routine appointments and lymphocyte subsets were requested as part of their clinical management.This was undertaken using our routine methodology, utilising BD instrumentation and BD Multitest™ IMK kit with BD Trucount™ tubes.After routine testing was completed, and results reported to the clinician, samples, considered waste material were used to evaluate the volumetric assay.Results were anonymised, and consent was not required.
Peripheral blood samples taken into tri-potassium EDTA (K3-EDTA) were tested using an established bead-based singleplatform flow cytometric method and the new single-platform volumetric approach.Samples that were seen to be haemolysed, lipaemic or icteric were excluded from the study.Samples were kept at ambient temperature until testing took place, with all samples tested within 48 h of venepuncture.Although the BD TBNK kit instructions for use recommends samples are tested within 24 h of venepuncture, in-house validation has demonstrated no statistically significant difference (where p < 0.05), between lymphocyte subset parameters measured at 0-24 h and at 24-48 h.All pipetting was performed by the same individual throughout the study using professionally serviced and calibrated pipettes and a reverse pipetting technique.An electronic pipette was used for blood and a manual pipette for antibodies and lysing reagents.
The BD Multitest™ 6-colour TBNK kit was used for the established bead-based assay.To undertake this, 20 μL of this TBNK reagent was added to a BD Trucount™ absolute counting tube, followed by 50 μL well-mixed blood.Tubes were vortexed and incubated for 15 min at ambient temperature in the dark.Following this, 450 μL BD FACS™ lysing reagent was then added, diluted 1 in 10 according to the manufacturer's instructions.The tubes were vortexed again and then incubated for a further 15 min at ambient temperature in the dark.These samples were then acquired and analysed on a BD FACSLyric™ flow cytometer using FACSuite™ Clinical software, version 1.4.
The volumetric assay was tested on the Sysmex XF-1600 flow cytometer using an EXBIO KOMBITEST™ 6 colour TBNK kit.The testing protocol for this instrument involved adding 20 μL of EXBIO TBNK reagent to a 12 mm Â 75 mm Falcon tube, followed by 50 μL of wellmixed blood.Samples were vortexed and incubated for 15 min at ambient temperature in the dark.The red cells were then lysed using 500 μL of a 1 in 10 dilution of Sysmex CyLyse™ FX, vortexed and incubated for a further 10 min at ambient temperature in the dark.These samples were then acquired and analysed on the Sysmex XF-1600 flow cytometer using XF-1600 IVD SW software, version 5.0.
Testing was undertaken to satisfy two requirements: to include a minimum of 100 total samples that met the study acceptance criteria and to ensure that a minimum of 30 samples with CD3 + CD4+ lymphocyte counts in the therapeutic range of 0-250 cells per microlitre (μL) were tested.

| Flow cytometry
Both technologies utilised automated software for sample acquisition and the sequence of gates used to derive the different lymphocyte subset populations were similar for both instruments.The lymphocytes were identified by CD45+ expression and low side scatter on a twodimensional dot plot and gated accordingly.A CD3 and side scatter dot plot of gated CD45+ lymphocyte events was used to distinguish between CD3+ and CD3-cells, with gates positioned around the CD3+ and CD3-lymphocytes.Further sequential gating of the CD3+ cells enabled CD4+ and CD8+ lymphocytes to be determined on a CD4 and CD8 dot plot.The CD3-lymphocytes were further classified on a CD19 and CD16/CD56 dot plot to derive CD19+ and CD16+/CD56+ lymphocytes.On the BD software, a further dot plot of CD19 and side scatter that included all events was utilised to identify the counting beads and a gate was positioned around the brightly fluorescent beads.The Sysmex software had an additional gate around the CD45+ WBCs on the CD45 and side scatter dot plot which is required to calculate absolute lymphocyte subset results.A side scatter time plot was displayed on the Sysmex software to detect any fluctuations in flow rate.
The FACSuite™ Clinical software on the BD FACSLyric™ performed automatic gate placement, highlighted spurious results that required review and possible re-positioning of gates and calculated absolute counts automatically.This was necessary in 21/118 cases.
The software for lymphocyte subset analysis on the Sysmex XF-1600 was a pre-release version that did not have the final, fully automated gating facility.The version that was used, displayed cellular events according to the template and gating sequence outlined earlier, however manual adjustment of gates was required.All calculations to determine the absolute cell counts were performed automatically by the software.All manual gate adjustments that were required were undertaken by the same experienced scientist on both instruments.

| Statistical analysis
For each platform, the absolute number of CD3+, CD3 + CD4+, CD3 + CD8+, CD19+ and CD3-CD16+/CD56+ lymphocytes were compared using Bland-Altman analysis and linear regression.The study set out to assess whether a volumetric, flow cytometric technique, using a metrologically traceable calibrant could achieve absolute lymphocyte subset results comparable to an established beadbased method.Therefore, although percentage values were obtained, the associated statistics have not been included in this report.
An additional analysis of CD3 + CD4+ lymphocyte counts less than 250 cells/μL was also undertaken to assess the bias at this lower cell count range.This cut-off was chosen as clinical treatment decisions for opportunistic infections in HIV patients are made on results falling below this range. 2sessment of bias was undertaken using Bland-Altman analysis, linear regression analysis was used to show the relationship between the two sets of results, and the Student's paired t-test to illustrate if there was a statistically significant difference.A p value of <0.05 was considered statistically significant.Statistics were calculated using Microsoft Excel.

| RESULTS
Of the 118 samples that were received for routine lymphocyte subsets and that met the acceptance criteria, 84 were from HIV+ patients, 21 were from patients undergoing monitoring for immune-related T A B L E 1 Summary of R 2 values for all absolute lymphocyte subset parameters and CD3 + CD4+ results in the range of 0-250 cells/μL.Regression analysis showed an excellent correlation between the two methods, with R 2 values for each parameter of > = 0.95.Table 1 summarises the linear regression R 2 values obtained for the absolute lymphocyte subset parameters, along with the CD3 + CD4+ results in the range of 0-250 cells/μL.Figure 1 illustrates the associated linear regression analysis plots for absolute lymphocyte subset parameters comparing Sysmex XF-1600 with BD FACSLyric.
Bland-Altman analysis for the absolute lymphocyte subset parameters indicated there was no significant bias (where p < 0.05) for absolute CD3 + CD4+ lymphocytes in the defined therapeutic range of 0-250 cells/μL (mean bias: 0.27 cells/μL), although positive biases were seen for CD3 + CD4+ lymphocytes (over the entire range tested: 14-1798 cells/μL) and CD3-CD16+/CD56+ lymphocytes (mean bias: 10.83 cells/μL and 6.79 cells/μL, respectively).Negative biases were seen for CD3 + CD8+ and CD19+ lymphocytes (mean bias: À29.17 cells/μL and À 18.76 cells/μL, respectively).Table 2 shows the mean differences obtained for the absolute counts, with 95% confidence intervals, ±1.96 s and associated p-values.established using the manufacturer's calibrator, to then generate single platform results for the lymphocyte subset populations.This calibrator has an assigned value for the WBC that is metrologically traceable back to an internationally recognised reference method according to the recommendations of ICSH guidelines. 25In principle this allows the flow cytometer absolute count results to be metrologically traceable.However, several factors may influence the results of A slight positive bias was observed for CD3 + CD4+ and CD3-CD16+/CD56+ results.Although statistically significant, the mean differences were deemed to be within clinically acceptable limits.Weak expression of CD16+/CD56+ with minimal separation between positive and negative populations was seen in some cases which may have contributed to the variation seen.
A slight negative bias was seen in both CD19+ and CD3 + CD8+ lymphocytes which was statistically significant, and occasional outlying results were observed.A retrospective analysis was undertaken to assess whether a positive/negative bias was apparent across all parameters, that might indicate a pipetting anomaly.This was apparent in two cases Time plots were examined on the Sysmex XF-1600 to check for an erratic flow rate but no discrepancies were observed.
Gate placement and separation between positive and negative populations were also re-reviewed.In several instances, it was difficult to differentiate the lymphocyte population from neighbouring cells on the Sysmex software due to many lymphocyte events acquired and the inability to enlarge the dot plots.Despite the outlying results, the overall mean bias was within clinically acceptable limits.We acknowledge, however, that absolute counts may not be comparable between different manufacturers' equipment and that a new reference range may be required if changing the method.The analysis software that was used on the XF-1600 required some manual adjustment but is still under development.For fully automated gating, without the need for manual intervention, an upgrade is necessary.
The results from this study suggest that as proof of concept, the calibration technique used on the Sysmex XF-1600 flow cytometer can produce results for lymphocyte subsets that are comparable to an established CE-IVD labelled 6-colour TBNK assay and may have the benefit of being metrologically traceable.Although to maintain an unbroken chain of calibration, automated gating which has not undergone manual intervention, would be required so as not to invalidate this claim.This is a positive step forward in flow cytometry, although it is recognised that further evaluation is necessary.

AUTHOR CONTRIBUTIONS
R Ward: Main Author, M Stevens: Sample testing and analysis, S Bashir: 2nd author contribution.

conditions and 9
were from patients who had undergone an allogeneic haemopoietic stem cell transplant.In addition, 1 patient had a slight lymphocytosis, 1 patient had undergone CAR T-Cell therapy and 2 specimens were from a UK NEQAS LI immune monitoring trial.

Figure 2 F
illustrates the associated Bland-Altman plots for the absolute lymphocyte subset parameters and CD3 + CD4+ lymphocytes with absolute counts in the range of 0-250 cells/μL.I G U R E 1 Regression analysis plots for absolute lymphocyte subset parameters (n = 118), comparing Sysmex XF-1600 with BD FACSLyric.(A) CD3+ cells, (B) CD3 + CD4+ cells, (C) CD3 + CD4+ cells in range of 0-250 cells/μL (n = 30), (D) CD3 + CD8+ cells, (E) CD3-CD16+/ CD56+ cells, (F) CD19+ cells.4| DISCUSSIONSysmex Corporation (Kobe, Japan) has recently developed the XF-1600 clinical flow cytometer that incorporates a volumetric method for the absolute counting of lymphocyte subsets.To achieve this, the WBC is measured during sample acquisition providing a single-platform analysis.The software algorithm that is used to calculate the absolute WBC (cells/μL) incorporates a calibration factor, T A B L E 2 Results of Bland-Altman analysis, summarizing mean bias with 95% confidence intervals, +/À1.96s and p-values for the absolute lymphocyte subsets parameters and CD3 + CD4+ lymphocytes with absolute counts in the range of 0 to 250 cells/μL.