Combined use of multiparametric flow cytometry and cytomorphology to enhance detection of neuroblastoma metastatic cells in bone marrow.

INTRODUCTION: In the context of neuroblastoma (NB), the screening for bone marrow (BM) metastasis is a recurrent issue for hematology laboratory routine practice. Detection of low tumor burden using light microscopy is often difficult. In this regard, our objective was to evaluate the performance of multiparametric flow cytometry (FC) for detecting NB metastatic cells in BM. METHODS: We applied a new FC multiparametric panel allowing the analysis of the co-expression of 5 surface markers: GD2 (disialoganglioside 2), CD9, CD56, CD81, and CD90, on CD45-negative BM cell populations, and compared results with BM biopsy immunohistochemistry, which is the reference method. RESULTS: In spike-in tests, the multiparametric FC successfully detected NB cells mixed in peripheral blood mononuclear cells to a level of 0.01%. FC analysis was performed on 45 sets of BM aspirates sampled from 21 children, either at diagnosis or during follow-up. Combining multiparametric FC with light microscopy improved NB metastasis detection, with a higher sensitivity (76.9% vs 61.5%) and a higher specificity (94.4% vs 77.8%) as compared to light microscopy alone. At the time of diagnosis, multiparametric FC detected NB metastatic cells in all cases. CONCLUSION: These results illustrate the performance of multiparametric FC analysis to detect metastatic BM infiltration of NB. This is of particular interest in an emergency context, since when combined with light microscopy, it enhances the detection of metastatic invasion within a short timeframe, allowing an adapted and rapid clinical management.

Standardization of Flow Cytometric Immunophenotyping for Hematological Malignancies: The FranceFlow Group Experience.

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry.

Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

Validation of a single-platform method for hematopoietic CD34+ stem cells enumeration according to accreditation procedure.

INTRODUCTION: Stem cells for autologous and allogenic transplantation are obtained from several sources including bone marrow, peripheral blood or cord blood. Accurate enumeration of viable CD34+ hematopoietic stem cells (HSC) is routinely used in clinical settings, especially to monitor progenitor cell mobilization and apheresis. The number of viable CD34+ HSC has also been shown to be the most critical factor in haematopoietic engraftment. The International Society for Cellular Therapy actually recommends the use of single-platform flow cytometry system using 7-AAD as a viability dye. AIM: In a way to move routine analysis from a BD FACSCaliburTM instrument to a BD FACSCantoTM II, according to ISO 15189 standard guidelines, we define laboratory performance data of the BDTM Stem Cell Enumeration (SCE) kit on a CE-IVD system including a BD FACSCanto II flow cytometer and the BD FACSCantoTM Clinical Software. InterQCTM software, a real time internet laboratory QC management system developed by VitroTM and distributed by Becton DickinsonTM, was also tested to monitor daily QC data, to define the internal laboratory statistics and to compare them to external laboratories. METHODS: Precision was evaluated with BDTM Stem Cell Control (high and low) results and the InterQC software, an internet laboratory QC management system by Vitro. This last one drew Levey-Jennings curves and generated numeral statistical parameters allowing detection of potential changes in the system performances as well as interlaboratory comparisons. Repeatability, linearity and lower limits of detection were obtained with routine samples from different origins. Agreement evaluation between BD FACSCanto II system versus BD FACSCalibur system was tested on fresh peripheral blood, freeze-thawed apheresis, fresh bone marrow and fresh cord blood samples. RESULTS: Instrument's measure and staining repeatability clearly evidenced acceptable variability on the different samples tested. Intra- and inter-laboratory CV in CD34+ cell absolute count are consistent and reproducible. Linearity analysis, established between 2 and 329 cells/µl showed a linear relation between expected counts and measured counts (R2=0.97). Linear regression and Bland-Altman representations showed an excellent correlation on samples from different sources between the two systems and allowed the transfer of routine analysis from BD FACSCalibur to BD FACSCanto II. CONCLUSIONS: The BD SCE kit provides an accurate measure of the CD34 HSC, and can be used in daily routine to optimize the enumeration of hematopoietic CD34+ stem cells by flow cytometry. Moreover, the InterQC system seems to be a very useful tool for laboratory daily quality monitoring and thus for accreditation.

Development of a new method for identification and quantification in cerebrospinal fluid of malignant cells from breast carcinoma leptomeningeal metastasis.

BACKGROUND: The diagnosis of leptomeningeal metastasis (LM) in patients with solid tumors remains difficult. The usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI of the entire neuraxis lack both specificity and sensitivity. The Veridex CellSearch® technology has been designed for the detection of circulating tumor cells (CTC) in blood from cancer patients and validated for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer. Our aim was to adapt this technology for the detection and the enumeration of tumor cells in the CSF of breast cancer patients presenting with LM. METHODS: On the occasion of a randomized phase III study evaluating the role of the intrathecal treatment in LM from breast cancer (DEPOSEIN, EudraCT N°: 2010-023134-23), the CellSearch® technology was adapted to direct enrichment, enumeration and visualization of tumor cells in 5 mL CSF samples, collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling. RESULTS: Sixteen CSF of 8 patients with primary breast cancer presenting with LM were studied. EpCAM+/cytokeratin + cells with typical morphology could be observed and enumerated sequentially with reproducible results in low or elevated numbers in 8 patients. CONCLUSION: This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of cancer patients with LM, especially to appreciate the efficacy of chemotherapy.

Increased gingival crevicular fluid levels of soluble triggering receptor expressed on myeloid cells (sTREM) -1 in severe periodontitis.

AIM: This study was designed to evaluate the presence of a new regulator of innate immunity in periodontitis: the soluble form of triggering receptor on myeloid cells-1 (sTREM-1) in gingival crevicular fluid (GCF). MATERIAL AND METHODS: GCF was collected at four sites, three pathological and one healthy from 17 patients with periodontitis, and at one healthy site from 23 control patients. An enzyme-linked immunosorbent assay (ELISA) kit was used to quantify sTREM-1 levels in collected crevicular fluid. Recorded clinical parameters were probing pocket depth (PPD), bleeding upon probing, tooth mobility, plaque index (PlI), and gingival index (GI). RESULTS: The mean sTREM-1 level in collected fluid was significantly higher in pathological sites than in healthy sites from either periodontal or control patients: 353.9 pg/ml, 50.2 pg/ml and 25.4 pg/ml respectively. Soluble TREM-1 concentration was significantly correlated with PPD. The sTREM-1 levels increased with the augmentation of the PlI and GI scores and levelled off at score 2 for both indexes. In multivariate analysis, periodontal pocket depth and smoking status were statistically associated with highest sTREM-1 concentrations. CONCLUSION: sTREM-1 was detected in crevicular fluid and its concentration was higher in pathological sites. It could be a marker of periodontal tissue destruction.

Soluble TREM-like transcript-1 regulates leukocyte activation and controls microbial sepsis.

The triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role during the onset of sepsis by amplifying the host immune response. The TREM-like transcript-1 (TLT-1) belongs to the TREM family, is selectively expressed on activated platelets, and is known to facilitate platelet aggregation through binding to fibrinogen. In this study, we show that a soluble form of TLT-1 is implicated in the regulation of inflammation during sepsis by dampening leukocyte activation and modulating platelet-neutrophil crosstalk. A 17-aa sequence of the TLT-1 extracellular domain (LR17) is responsible for this activity through competition with the TREM-1 ligand. Whereas early or late LR17 treatment of septic mice improves survival, treml-1(-/-) animals are highly susceptible to polymicrobial infection. The present findings identify platelet-derived soluble TLT-1 as a potent endogenous regulator of sepsis-associated inflammation and open new therapeutic perspectives. We anticipate soluble TLT-1 to be important in regulating leukocyte activation during other noninfectious inflammatory disorders.

Combination biomarkers to diagnose sepsis in the critically ill patient.

RATIONALE: Although the outcome of sepsis benefits from the prompt administration of appropriate antibiotics on correct diagnosis, the assessment of infection in critically ill patients is often a challenge for clinicians. In this setting, simple biomarkers, especially when used in combination, could prove useful. OBJECTIVES: To determine the usefulness of combination biomarkers to diagnose sepsis. METHODS: Three hundred consecutive patients were enrolled to construct a biologic score that was next validated in an independent prospective cohort of 79 critically ill patients from another center. MEASUREMENT AND MAIN RESULTS: Plasma concentrations of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and procalcitonin (PCT) were assayed, and the expression of the high-affinity immunoglobulin-Fc fragment receptor I (FcγRI) CD64 on neutrophils (polymorphonuclear [PMN] CD64 index) in flow cytometry was measured. A "bioscore" combining these biomarkers was constructed. Serum concentrations of PCT and sTREM-1 and the PMN CD64 index were higher in patients with sepsis compared with all others (P < 0.001 for the three markers). These biomarkers were all independent predictors of infection, the best receiver-operating characteristic curve being obtained for the PMN CD64 index. The performance of the bioscore, better than that of each individual biomarker, was externally confirmed in the validation cohort. CONCLUSIONS: This prospective study, including inceptive and validation cohorts of unselected intensive care unit patients, demonstrates the high performance of a bioscore combining the PMN CD64 index together with PCT and sTREM-1 serum levels in diagnosing sepsis in the critically ill patient.

Plasma soluble triggering receptor expressed on myeloid cells-1 in Crohn's disease.

BACKGROUND: No definite conclusions can be drawn from available data on the accuracy of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) to assess disease activity in Crohn's disease. AIMS: Plasma sTREM-1 levels were correlated with disease activity markers in Crohn's disease. METHODS: 191 consecutive patients from a single referral centre (Nancy IBD cohort) were prospectively enrolled between June 1, 2005 and December 12, 2008. Plasma sTREM-1 levels were also assessed amongst 20 healthy controls. RESULTS: The sTREM-1 was detectable in 87 Crohn's disease patients (46%). Plasma sTREM-1 level was higher in Crohn's disease patients (interquartile range, 0-356) than in healthy controls (interquartile range, 0-15.1; P=0.01). It was neither correlated with Crohn's disease activity index (r=0.05, P=0.56), C-reactive protein (r=0.06, P=0.53), nor with albumin (r=-0.041, P=0.66). Crohn's disease activity index, C-reactive protein and albumin median levels were similar between patients with positive sTREM-1 levels and those with undetectable sTREM-1 levels. Azathioprine (P=0.06), infliximab (P=0.68) and methotrexate (P=0.56) did not influence sTREM-1 levels. CONCLUSION: Plasma sTREM-1 does not appear to be an accurate marker of disease activity in Crohn's disease and cannot be recommended for assessing disease activity in these patients.

Serum sTREM-1 (soluble triggering receptor expressed on myeloid cells-1) associates negatively with embryo quality in infertility patients.

PROBLEM: Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a useful biomarker of infection and inflammation. METHOD OF STUDY: We studied serum and follicular fluid sTREM-1 in infertile patients (N = 110) utilizing enzyme-linked immunosorbent assay. RESULTS: Serum and follicular sTREM-1 were in good correlation (Pearson's correlation 0.56, P < 0.0001) with higher values in follicular fluid (140.4 ± 34.4 and 115.6 ± 35.1 pg/mL, t-test, P < 0.0001). Endometriosis associated with lower follicular and serum sTREM-1 compared with male factor infertility patients (age-adjusted r = -25.7 pg/mL, P = 0.018; r = -22.1 pg/mL, P = 0.030). No associations between follicular or serum sTREM-1 and clinical parameters were found, except higher serum sTREM-1 associated with lower embryo quality in all patients (adjusted r = -0.3%, P = 0.033), with a cutoff value between 111.5 and 113.3 pg/mL (OR = 0.38, P = 0.048; OR = 0.34, P = 0.028) predicting that more than 39% of embryos would be with good quality. CONCLUSION: Serum sTREM-1 could represent a prognostic marker for female fecundity, probably indicating impaired inflammatory reaction of immune system.