Assessment of the AQUIOS flow cytometer - An automated sample preparation system for CD4 lymphocyte PanLeucogating enumeration.

BACKGROUND: Flow cytometry has been the approach of choice for enumerating and documenting CD4-cell decline in HIV monitoring. Beckman Coulter has developed a single platform test for CD4+ T-cell lymphocyte count and percentage using PanLeucogating (PLG) technology on the automated AQUIOS flow cytometer (AQUIOS PLG). OBJECTIVES: This study compared the performance of AQUIOS PLG with the Flowcare PLG method and performed a reference interval for comparison with those previously published. METHODS: The study was conducted between November 2014 and March 2015 at 5 different centres located in Canada; Paris, France; Lyon, France; the United States; and South Africa. Two-hundred and forty samples from HIV-positive adult and paediatric patients were used to compare the performances of AQUIOS PLG and Flowcare PLG on a FC500 flow cytometer (Flowcare PLG) in determining CD4+ absolute count and percentage. A reference interval was determined using 155 samples from healthy, non-HIV adults. Workflow was investigated testing 440 samples over 5 days. RESULTS: Mean absolute and relative count bias between AQUIOS PLG and Flowcare PLG was -41 cells/µL and -7.8%. Upward and downward misclassification at various CD4 thresholds was ≤ 2.4% and ≤ 11.1%. The 95% reference interval (2.5th - 97.5th) for the CD4+ count was 453-1534 cells/µL and the percentage was 30.5% - 63.4%. The workflow showed an average number of HIV samples tested as 17.5 per hour or 122.5 per 8-hour shift for one technician, including passing quality controls. CONCLUSION: The AQUIOS PLG merges desirable aspects from conventional flow cytometer systems (high throughput, precision and accuracy, external quality assessment compatibility) with low technical operating skill requirements for automated, single platform systems.

Implementation of an NGS-based sequencing and gene fusion panel for clinical screening of patients with suspected hematologic malignancies.

OBJECTIVES: The diagnosis of hematologic malignancies integrates multiple diagnostic and clinical disciplines. Historically, targeted (single-analyte) genetic testing has been used as reflex to initial prescreening by other diagnostic modalities including flow cytometry, anatomic pathology, and clinical cytogenetics. Given the wide range of mutations associated with hematologic malignancies a DNA/RNA-based NGS panel can provide a more effective and economical approach to comprehensive testing of patients as an initial, tier-1 screen. METHODS: Using a cohort of 380 patients, we performed clinical validation of a gene panel designed to assess 40 genes (DNA), and 29 fusion driver genes with over 600 gene fusion partners (RNA), including sample exchange data across three clinical laboratories, and correlation with cytogenetic testing results. RESULTS: The clinical validation of this technology demonstrated that its accuracy, sensitivity, and specificity are comparable to the majority of targeted single-gene approaches, while assessment of the initial patient cohort data demonstrated a high diagnostic yield of 50.5%. CONCLUSIONS: Implementation of a tier-1 NGS-based protocol for gene panel screening provides a comprehensive alternative to targeted molecular testing in patients with suspected hematologic malignancies, with increased diagnostic yield, scalability, reproducibility, and cost effectiveness, making it ideally suited for implementation in clinical laboratories.

Paroxysmal Nocturnal Hemoglobinuria Assessment by Flow Cytometric Analysis.

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon but frequently debilitating disease that, if untreated, may lead to death in up to 35% of patients within 5 years. Assessment of PNH clone size by flow cytometric analysis has increased in importance with the availability of therapeutic treatments, which prevent the hemolysis of red blood cells and, hence, the myriad symptoms that accompany the disease. This article addresses flow cytometric methodologies and highlights areas of importance in implementing testing, not only for classic PNH but also for other related bone marrow failure disorders, such as aplastic anemia and low-grade myelodysplastic syndrome.

Determination of optimal replicate number for validation of imprecision using fluorescence cell-based assays: proposed practical method.

BACKGROUND: Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell-based fluorescence assays performed on flow cytometers are currently lacking. METHODS: Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined. RESULTS: For all assays and most instrument platforms, <5 replicates were found adequate to validate assay imprecision levels below the 5-10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95% CI and the cumulative mean for replicates, termed variance factor (VF), provide a data-driven means for determining optimal replicate numbers. CONCLUSIONS: The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate that three to four replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes.

Determination of optimal replicate number for validation of imprecision using fluorescence cell based assays: Proposed practical method.

Background: Assay validation includes determination of inherent imprecision across the reportable range. However specific practical guidelines for determinations of precision for cell based fluorescence assays performed on flow cytometers are currently lacking. Methods: Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical IVD use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 (hENT1) quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell (HSC) enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals of the mean (95%CI), standard deviation and coefficient of variation (CV) of sequential replicates were determined. Results: For all assays and most instrument platforms <5 replicates were found adequate to validate assay imprecision levels below the 5-10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95%CI and the cumulative mean for replicates, termed variance factor (VF), provide a data driven means for determining optimal replicate numbers. Conclusions: The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate 3-4 replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes. © 2013 Clinical Cytometry Society.

User-defined protein marker assay development for characterization of circulating tumor cells using the CellSearch® system.

The majority of cancer-related deaths result from metastasis, which has been associated with the presence of circulating tumor cells (CTCs). It has been shown that CTC cut-off values exist that predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. Development of the CellSearch® system (Veridex) has allowed for sensitive enumeration of CTCs. In the current study, protocols were developed and optimized for use with the CellSearch system to characterize CTCs with respect to user-defined protein markers of interest in human blood samples, including the cancer stem cell marker CD44 and the apoptosis marker M-30. Flow cytometry (FCM) experiments were initially carried out to assess expression of CD44 and M-30 on MDA-MB-468 human tumor cells. Human blood samples were then spiked with MDA-MB-468 cells and processed with the appropriate antibody (CD44/M-30) on the CellSearch. Detailed optimization of CD44 was carried out on the CellSearch using various antibody concentrations, exposure times, and cell lines with varying CD44 expression. Troubleshooting experiments were undertaken to explain observed discrepancies between FCM and CellSearch results for the M-30 marker. After extensive optimization, the best CD44/M-30 concentrations and exposure times were determined to be 1.5/3.5 µg/ml and 0.2/0.8 s, respectively. The percentage of CD44(+) tumor cells was 99.5 ± 0.39% by FCM and 98.8 ± 0.51% by the CellSearch system. The percentage of M-30(+) tumor cells following paclitaxel treatment was 17.6 ± 1.18% by FCM and 10.9 ± 2.41% by CellSearch. Proper optimization of the CD44 marker was achieved; however, M-30 does not appear to be a suitable marker for use in this platform. Taken together, the current study provides a detailed description of the process of user-defined protein marker development and optimization using the CellSearch, and will be an important resource for the future development of protein marker assays by users of this platform.

Elevated activated partial thromboplastin time does not correlate with heparin rebound following cardiac surgery.

PURPOSE: Exposure to cardiopulmonary bypass (CPB) is associated with postoperative coagulopathy and hemorrhage. Recent literature indicates that heparin rebound occurs almost universally following cardiac surgery. We conducted this pilot study to evaluate if the presence of residual circulating heparin following cardiac surgery can be diagnosed by elevation of activated partial thromboplastin time (APTT). METHOD: After obtaining Research Ethics Board approval, blood samples from 30 patients receiving heparin for CPB were evaluated at the time of intensive care unit admission and 2, 4, and 6 hr thereafter. Activated clotting time, whole blood heparin concentration (Hepcon HMS Plus, Medtronic), anti-Xa levels, and APTT were measured at each time point. Samples with prolonged APTT were subjected to mechanistic studies with heparin adsorption and 1:1 mixing. RESULTS: Anti-Xa was elevated in 52 of the 120 blood samples (0.08 +/- 0.08 U . mL(-1), mean +/- SD). APTT was elevated in 49 (40.8%) samples with an average of 51.4 +/- 31.9 sec. At all time points, the APTT correlated poorly with anti-Xa levels with correlation coefficients ranging from -0.26 to -0.05. Mean APTT was modestly, but not significantly, associated with total dose of protamine with r = 0.34 (CI: -0.03, 0.62). After 1:1 mixing studies, APTT returned to normal in most (82%) samples tested. CONCLUSION: Circulating residual heparin is commonly presented following cardiac surgery and does not correlate with APTT. Considering that mixing studies normalize APTT in most samples, elevated APTT following CPB may reflect deficiency of coagulation factors or presence of a coagulation inhibitor such as protamine. Further studies are required to confirm this observation.

Treatment of acquired factor X inhibitor by plasma exchange with concomitant intravenous immunoglobulin and corticosteroids.

A patient with spontaneous hemorrhage from multiple body sites was found to have markedly prolonged international normalized ratio (INR) and activated partial thromboplastin times (aPTT) with incomplete correction of aPTT on mixing studies using normal plasma. The cause of this severe hemorrhage was due to a specific factor X inhibitor. No underlying or associated diseases were found. Initial treatment with fresh frozen plasma, vitamin K, and recombinant activated factor VII (rFVIIa) was unsuccessful. However, therapy utilizing plasma exchange with concomitant intravenous immunoglobulin and corticosteroids resulted in a rapid and sustained normalization of factor X levels with a clinical hemostatic response.

An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia.

BACKGROUND: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. METHODS: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. RESULTS: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. CONCLUSIONS: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory.

Reduction of variation in T-cell subset enumeration among 55 laboratories using single-platform, three or four-color flow cytometry based on CD45 and SSC-based gating of lymphocytes.

BACKGROUND: Enumeration of CD4(+) and CD8(+) T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxembourg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for > or =75% of the participants. METHODS: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. RESULTS: With the standard technique, between-site CVs of approximately 8% (CD3+ T cells), approximately 9% (CD4+ T cells), and approximately 10% (CD8+ T cells) were achieved. Within-site CVs were <5% for 82% (CD3+ T cells) and approximately 70% (CD4+ and CD8+ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3+, CD4+, and CD8+ T cells. CONCLUSIONS: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3+, CD4+, and CD8+ T-cell counts.