Cytometry Part B: Clinical Cytometry最新文献

Rare acute leukemia (AL) components or subtypes such as blastic plasmacytoid dendritic cell neoplasm (BPDCN) or early T-cell precursor acute Lymphoblastic Leukemia (ETP-ALL) can be difficult to detect by routine flow cytometry due to their immunophenotypes overlapping with other poorly differentiated AL. We hypothesized that using standardized EuroFlow™ Consortium approach could better diagnose such entities among cases that previously classified as acute myeloid leukemia (AML)-M0, AML with minimal differentiation, AML with myelodysplasia-related changes without further lineage differentiation, and AL of ambiguous lineage. In order to confirm this hypothesis and assess whether these AL subtypes such as BPDCN and ETP-ALL had previously gone undetected, we reanalyzed 49 banked cryopreserved sample cases using standardized EuroFlow™ Consortium panels. We also performed target sequencing to capture the mutational commonalities between these AL subtypes. Reanalysis led to revised or refined diagnoses for 23 cases (47%). Of these, five diagnoses were modified, uncovering 3 ETP-ALL and 2 typical BPDCN cases. In 12 AML cases, a variable proportion of immature plasmacytoid dendritic cell and/or monocytic component was newly identified. In one AML case, we have identified a megakaryoblastic differentiation. Finally, in five acute lymphoblastic leukemia (ALL) cases, we were able to more precisely determine the maturation stage. The application of standardized EuroFlow flow cytometry immunophenotyping improves the diagnostic accuracy of ALs and could impact treatment decisions.

Cervical cancer (CC) is the fourth most common malignant tumor in women worldwide. Detecting different biomarkers together on single cells by novel method mass cytometry could contribute to more precise screening. Liquid-based cytology (LBC) cervical samples were collected (N = 53) from women categorized as normal and precancerous lesions. Human papillomavirus was genotyped by polymerase chain reaction, while simultaneous examination of the expression of 29 proteins was done by mass cytometry (CyTOF). Differences in cluster abundances were assessed with Spearman's rank correlation as well as high dimensional data analysis (t-SNE, FlowSOM). Cytokeratin (ITGA6, Ck5, Ck10/13, Ck14, Ck7) expression patterns allowed determining the presence of different cells in the cervical epithelium. FlowSOM analysis enabled to phenotype cervical cells in five different metaclusters and find new markers that could be important in CC screening. The markers Ck18, Ck18, and CD63 (Metacluster 3) showed significantly increasing associated with severity of the precancerous lesions (Spearman rank correlation rho 0.304, p = 0.0271), while CD71, KLF4, LRIG1, E-cadherin, Nanog and p53 (Metacluster 1) decreased with severity of the precancerous lesions (Spearman rank correlation rho −0.401, p = 0.0029). Other metaclusters did not show significant correlation, but metacluster 2 (Ck17, MCM, MMP7, CD29, E-cadherin, Nanog, p53) showed higher abundance in low- and high-grade intraepithelial lesion cases. CyTOF appears feasible and should be considered when examining novel biomarkers on cervical LBC samples. This study enabled us to characterize different cells in the cervical epithelium and find markers and populations that could distinguish precancerous lesions.

Automated analysis of flow cytometry data can improve objectivity and reduce analysis time but has generally required work by software and algorithm experts. Here, we investigated the performance of BD ElastiGate™ Software (hereafter ElastiGate), which allows users to automate gating by selecting gated training files, then uses elastic image registration to gate new files. Three assays of increasing complexity were examined: TBNK, stem cell enumeration (SCE), and lymphoid screening tube (LST). For TBNK analysis, 60 peripheral blood (PB) samples from normal, HIV+, and controls were tested with ground truth analysis by an existing automated method. For SCE, 128 samples including bone marrow (BM), cord blood (CB), and apheresis were tested with analysis by multiple manual analysts. For LST, 80 PB and 28 BM samples were tested with manual analysis. For ElastiGate, a minimal number of training files was selected. Results were compared by Bland–Altman or F1 score analysis. For TBNK, ElastiGate using three training files (1 control, 1 normal, 1 HIV+) showed mean %bias across all reported populations between −1.48% and 7.13% (average 2.08%). For SCE, ElastiGate using three BM and two CB training files showed median F1 scores >0.93 in comparison to >0.94 and >0.92 for two other manual analysts. For LST, ElastiGate using four training files for each of PB and BM showed median F1 scores >0.945 for 13 of 14 PB populations and 10 of 14 BM populations, with generally similar or better performance for normal samples compared to abnormal; populations with lower scores were often associated with lower agreement between manual analysts. Based on analysis of three assays with four sample types of increasing complexity, ElastiGate with minimal training files may perform as an automated gating assistant. The results reported here are for research use only, not for use in diagnostic or therapeutic procedures.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymopathy that affects red blood cells (RBCs) and renders them susceptible to oxidative stress. G6PD deficiency can cause hemolytic anemia, especially after exposure to certain drugs or infections. The diagnosis of G6PD deficiency is usually based on spectrophotometric measurement of enzyme activity, but this method has limitations in heterozygous females and in patients with other hematological disorders. In this study, we evaluated the use of flow cytometry as an alternative method for detecting G6PD deficiency in 514 samples (265 females and 249 males) from a clinical laboratory. We compared the results of flow cytometry with those of spectrophotometry and molecular analysis, and assessed the performance of flow cytometry in different subgroups of patients. We found that flow cytometry was able to identify G6PD deficiency in most cases, with high sensitivity and specificity. Flow cytometry also allowed the quantification of the percentage of G6PD-deficient RBCs, which varied among heterozygous females due to X-chromosome inactivation. Moreover, flow cytometry detected several cases of G6PD deficiency that were missed by spectrophotometry, especially in heterozygous females with normal or subnormal enzyme activity. However, flow cytometry also showed some false negative results, mainly in patients with sickle cell disease. Therefore, flow cytometry is a reliable and efficient tool for screening G6PD deficiency, but some precautions should be taken in interpreting the results in patients with other hematological conditions.

High-grade B-cell lymphomas (HGBCL) represent a heterogeneous group of very rare mature B-cell lymphomas. The 4th revised edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues (WHO-HAEM) previously defined two categories of HGBCL: the so-called double-hit (DHL) and triple-hit (THL) lymphomas, which were related to forms harboring MYC and BCL2 and/or BCL6 rearrangements, and HGBCL, NOS (not otherwise specified), corresponding to entities with intermediate characteristics between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL), without rearrangement of the MYC and BCL2, and/or BCL6 genes. In the 5th edition of the WHO-HAEM, DHL with MYC and BCL2 rearrangements or THL were reassigned as DLBCL/HGBCL with MYC and BCL2 rearrangements (DLBCL/HGBL-MYC/BCL2), whereas the category HGBCL, NOS remains unchanged. Characterized by an aggressive clinical presentation and a poor prognosis, HGBCL is often diagnosed at an advanced, widespread stage, leading to potential disseminated forms with a leukemic presentation, or spreading to the bone marrow (BM) or other biological fluids. Flow cytometric immunophenotypic study of these disseminated cells can provide a rapid method to identify HGBCL. However, due to the scarcity of cases, only limited data about the immunophenotypic features of HGBCL by multiparametric flow cytometry are available. In addition, identification of HGBCL cells by this technique may be challenging due to clinical, pathological, and biological features that can overlap with other distinct lymphoid malignancies, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and even B acute lymphoblastic leukemia (B-ALL). In this study, we aimed to characterize the detailed immunophenotypic portrait of HGBCL, evaluating by multiparametric flow cytometry (MFC) the expression of 26 markers on biological samples obtained from a cohort of 10 newly-diagnosed cases and comparing their level of expression with normal peripheral blood (PB) B lymphocytes (n = 10 samples), tumoral cells from patients diagnosed with B-ALL (n = 30), BL (n = 13), or DLBCL (n = 22). We then proposed a new and simple approach to rapidly distinguish disseminated forms of HGBCL, BL, and DLBCL, using the combination of MFC data for CD38, BCL2, and CD39, the three most discriminative markers explored in this study. We finally confirmed the utility of the scoring system previously proposed by Khanlari to distinguish HGBCL cells from B lymphoblasts of B-ALL. In conclusion, we described a distinct immunophenotypic portrait of HGBCL cells and proposed a strategy to differentiate these cells from other aggressive B lymphoma entities in biological samples.

HLA-B27 is a major histocompatibility complex (MHC) class I antigen which exhibits strong association (90%) with ankylosing spondylitis. HLA-B27 detection in patients by flow cytometry is a widely used clinical test, performed on many different flow cytometer models. We sought to develop and validate a test conversion protocol for the HLA-B27 test performed on the BD FACSCanto to BD's newer FACSLyric flow cytometers. The development and validation experiments were performed using anti-HLA-B27*FITC/CD3*PE antibody-stained whole blood patient specimens. The anti-HLA-B27*FITC logarithmic median fluorescence (LMF) results on the BD FACSCanto were converted to median fluorescence intensity (MFI) values on the BD FACSLyric. Clustering of the HLA-B27 positive and negative values, using a 3rd order polynomial equation, resulted in a conversion of the BD FACSCanto cutoff values, negative (<150 LMF) and positive (≥160 LMF), to negative (<4530 MFI) and positive (≥6950 MFI) on the BD FACSLyric. Accuracy was assessed by comparing the flow results obtained on the BD FACSCanto and BD FACSLyric to a molecular PCR based assay. Additional validation parameters (compensation verification, intra- and inter-assay precision, and instrument comparison) were performed per the recommendations outlined in the Clinical and Laboratory Standards Institute (CLSI) H62 guidelines for validation of flow cytometry assays.

The gut-liver axis includes the bidirectional communication between the gut and the liver, and thus covers signals from liver-to-gut and from gut-to-liver. Disruptions of the gut-liver axis have been associated with the progression of chronic liver diseases, including alcohol-related and metabolic dysfunction-associated steatotic liver disease and cholangiopathies. Immune cells and their expression of pattern recognition receptors, activation markers or immune checkpoints might play an active role in the communication between gut and liver. Here, we present a 26-color full spectrum flow cytometry panel for human cells to decipher the role of circulating immune cells in gut-liver communication during the progression of chronic liver diseases in a non-invasive manner, which has been optimized to be used on patient-derived whole blood samples, the most abundantly available clinical material. Our panel focuses on changes in pattern recognition receptors, including toll-like receptors (TLRs) or Dectin-1, and also includes other immunomodulatory molecules such as bile acid receptors and checkpoint molecules. Moreover, this panel can be utilized to follow the progression of chronic liver diseases and could be used as a tool to evaluate the efficiency of therapeutic targets directed against microbial mediators or modulating immune cell activation.