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

INTRODUCTION: 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. METHOD: A total of 118 samples received for lymphocyte subset analysis were tested using an established bead-based technique (BD Multitest™ 6-colour TBNK assay using Trucount™ tubes on a BD FACSLyric flow cytometer), followed by a volumetric method on the Sysmex XF-1600 flow cytometer using Exbio Kombitest 6-colour TBNK reagent. All samples were tested in accordance with the manufacturer's instructions. RESULTS: Absolute count values from both methodologies for CD3+, CD3 + CD4+, CD3 + CD8+, CD19+ and CD3-CD16+/CD56+ lymphocyte populations were compared using linear regression (R2 for all parameters >0.95) and Bland-Altman analysis. 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: -29.17 cells/µL and - 18.76 cells/µL, respectively). CONCLUSION: A high degree of correlation was found for results from both methodologies and observed bias was within the limits of clinical acceptability for all populations. This shows that the metrologically traceable lymphocyte subset absolute counts produced by the Sysmex XF-1600 are robust within clinically required limits.

Non-Responsive and Refractory Coeliac Disease: Experience from the NHS England National Centre.

We characterised the aetiology of non-responsive coeliac disease (NRCD) and provided contemporary mortality data in refractory coeliac disease (RCD) from our centre. We also measured urine gluten immunogenic peptides (GIPs) in patients with established RCD1 to evaluate gluten exposure in these individuals. METHODS: This was a longitudinal cohort study conducted in Sheffield, UK. Between 1998 and 2019, we evaluated 285 adult (≥16 years) patients with NRCD or RCD. Patients with established RCD1 and persisting mucosal inflammation and/or ongoing symptoms provided three urine samples for GIP analysis. RESULTS: The most common cause of NRCD across the cohort was gluten exposure (72/285; 25.3%). RCD accounted for 65/285 patients (22.8%), 54/65 patients (83.1%) had RCD1 and 11/65 patients (16.9%) had RCD2. The estimated 5-year survival was 90% for RCD1 and 58% for RCD2 (p = 0.016). A total of 36/54 (66.7%) patients with RCD1 underwent urinary GIP testing and 17/36 (47.2%) had at least one positive urinary GIP test. CONCLUSION: The contemporary mortality data in RCD2 remains poor; patients with suspected RCD2 should be referred to a recognised national centre for consideration of novel therapies. The high frequency of urinary GIP positivity suggests that gluten exposure may be common in RCD1; further studies with matched controls are warranted to assess this further.

Interlaboratory comparison of the TransFix®/EDTA Vacuum Blood Collection tube with the 5 mL Cyto-Chex® BCT.

BACKGROUND: An interlaboratory study was performed to compare the performance of the 3 mL TransFix®/EDTA Vacuum Blood Collection Tube (TVT), with the 5 mL Cyto-Chex® BCT tube (BCT). Both devices are intended for collection and storage of whole blood specimens for immunophenotyping of leukocytes by flow cytometry for up to 14 days. METHODS: One site in the United States and two in the United Kingdom tested samples from 10 HIV positive patients and four healthy subjects for a total of 42 samples. From each subject, three blood samples were collected: a BD 4 mL K3 EDTA Vacutainer (Vacutainer), a TVT, and a BCT. At all sites, samples were analyzed on a BD FACS Canto II flow cytometer for a full lymphocyte subset count within 6 h of collection (all devices) and on Day 11 and Day 15 (TVT and BCT only). Data obtained from the Vacutainer were used as the control data set with which TVT and BCT data were statistically compared. RESULTS AND CONCLUSIONS: Statistical concordance was demonstrated for both devices in relation to cell absolute count recovery. For cell marker signal, both devices exhibited a significant decrease in mean fluorescence intensity (MFI) for the detection of lymphocyte subsets and their target markers. There was a marked increase in autofluorescence observed for BCT stabilized lymphocytes whereas values for the TVTs were comparable to the control. There were eight instances of statistical equivalence between the level of antibody autofluorescence observed in the control tube and the TVT across both patient cohorts, versus two for the BCT. © 2018 International Clinical Cytometry Society.

Perfect count: a novel approach for the single platform enumeration of absolute CD4+ T-lymphocytes.

BACKGROUND: The derivation of reliable CD4(+) T lymphocyte counts is vital for the monitoring of disease progression and therapeutic effectiveness in HIV(+) individuals. Flow cytometry has emerged as the method of choice for CD4(+) T lymphocyte enumeration, with single-platform technology, coupled with reference counting beads, fast becoming the "gold standard." However, although single-platform, bead-based, sample acquisition requires the ratio of beads to cells to remain unchanged, there is no available method, until recently, to monitor this. METHODS: Perfect Count beads have been developed to address this issue and to incorporate two bead populations, with different densities, to allow the detection of inadequate mixing. Comparison of the relative proportions of both beads with the manufacture's defined limits enables an internal QC check during sample acquisition. In this study, we have compared CD4(+) T lymphocyte counts, obtained from 104 HIV(+) patients, using TruCount beads with MultiSet software (defined as the predicated method) and the new Perfect Count beads, incorporating an in house sequential gating strategy. RESULTS: We have demonstrated an excellent degree of correlation between the predicate method and the Perfect Count system (r(2) = 0.9955; Bland Altman bias +27 CD4(+) T lymphocytes/microl). CONCLUSIONS: The Perfect Count system is a robust method for performing single platform absolute counts and has the added advantage of having internal QC checks. Such an approach enables the operator to identify potential problems during sample preparation, acquisition and analysis.