Cytometry Part B: Clinical Cytometry最新文献

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is an uncommon mature T-cell neoplasm occurring in patients with textured breast implants, typically after 7–10 years of exposure. Although cytopathologic or histopathologic assessment is considered the gold standard diagnostic method for BIA-ALCL, flow cytometry (FC)-based immunophenotyping is recommended as an adjunct test. However, the diagnostic efficacy of FC is not well reported. We reviewed 290 FC tests from breast implant pericapsular fluid and capsule tissue from 182 patients, including 16 patients with BIA-ALCL over a 6-year period, calculating diagnostic rates and test efficacy. FC showed an overall sensitivity of 75.9%, specificity of 100%, and negative and positive predictive values of 95.4% and 100%, respectively. Blinded expert review of false-negative cases identified diagnostic pitfalls, improving sensitivity to 96.6%. Fluid samples had better rates of adequate samples for FC testing compared with tissue samples. Paired with FC testing of operating room (OR)-acquired fluid samples, capsulectomy FC specimens added no diagnostic value in patients with concurrent fluid samples; no cases had positive capsule FC with negative fluid FC. Fluid samples are adequate for FC testing more often than tissue. Capsule tissue FC specimens do not improve FC efficacy when paired with OR-acquired fluid FC samples and are often inadequate samples. FC is 100% specific for BIA-ALCL and can serve as a confirmatory test but should not be the sole diagnostic method. Awareness of sample-specific diagnostic pitfalls greatly improves the sensitivity of BIA-ALCL testing by FC.

Immunophenotyping by flow cytometry is an integral part of the diagnosis and classification of leukemias/lymphomas. The expression of ROR1 associated with chronic B lymphocytic leukemia (CLL) is well described in the literature, both in its diagnosis and in the follow-up of minimal residual disease (MRD) research, however, there are few studies regarding the expression pattern of ROR1 in other subtypes of mature B lymphoid neoplasms. With the aim of evaluating the expression of ROR1 and associating it with the expression of other important markers for the differentiation of mature B lymphoid neoplasms (MBLN), 767 samples of cases that entered our laboratory for immunophenotyping with clinical suspicion of MBLN were studied. ROR1 expression is predominant in CD5+/CD10− neoplasms. Overall, positive ROR1 expression was observed in 461 (60.1%) cases. The CD5+/CD10− group had a significantly higher proportion of ROR1 positive samples (89.9%) and more brightly expressed ROR1 than the other groups. Our results highlight the importance of evaluating ROR1 expression in the diagnosis of MBLN to contribute to the differential diagnosis, and possibly therapy of mainly CLL, and indicate that this marker could be considered as a useful addition to immunophenotypic panels, particularly for more challenging cases.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Since response to antigen-based immunotherapy relies upon the level of tumor antigen expression we developed an antigen quantification assay using ABC values. Antigen quantification as a clinical assay requires methods for quality control and for interlaboratory and inter-cytometer platform standardization. A single lot of Cytotrol™ Lyophilized Control Cells (Beckman Coulter) used for all studies. The variability in antigen quantification across 4 different instrument platforms in 2 separate laboratories was evaluated. The effect of the antibody clone utilized, importance of custom 1:1 molar ratio (fluorophore to protein, F/P) verses off-the-shelf antibodies, and QuantiBrite PE calibration verses linearity calibration combined with a single point scale transformation with CD4 as reference were determined. Use of single lot control cells allowed validation of reproducibility between flow cytometer platforms and laboratories and allowed assessment of different antibody lots, cocktail preparation, and different antibody clones. Off the shelf antibody preparations provide reproducible estimates of antigen density, however custom 1:1 unimolar antibody preparations should be utilized for definitive measurement of antigen expression.Geometric Mean fluorescent Intensity (GeoMFI) was not comparable across instruments and inter-laboratory. The use of CD4 as the reference marker can minimize variability in ABC values. Comparable antigen quantification is vital in managing patients receiving antigen-based immunotherapy. If this assay is to be utilized in a clinical setting, quality control methods have to be instituted to assure reproducibility and allow validation across laboratories. We have demonstrated that use of a lyophilized cell control is highly valuable in achieveing these goals.