A fully-standardized EuroFlow 8–color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia ...(ALL) patients with a sensitivity of ≤10−5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)–based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR–based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10−5), if sufficient cells (>4 × 106, preferably more) are evaluated.
•Standardized flow cytometry allows highly sensitive MRD measurements in virtually all BCP-ALL patients.•If sufficient cells are measured (>4 million), flow cytometric MRD analysis is at least as sensitive as current PCR-based MRD methods.
Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains ...poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring.
Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to ...the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis.
A subset of autoimmune diseases is characterized by predominant pathogenic IgG4 autoantibodies (IgG4-AID). Why IgG4 predominates in these disorders is unknown. We hypothesized that dysregulated B ...cell maturation or aberrant class switching causes overrepresentation of IgG4
B cells and plasma cells. Therefore, we compared the B cell compartment of patients from four different IgG4-AID with two IgG1-3-AID and healthy donors, using flow cytometry. Relative subset abundance at all maturation stages was normal, except for a, possibly treatment-related, reduction in immature and naïve CD5
cells. IgG4
B cell and plasma cell numbers were normal in IgG4-AID patients, however they had a (sub)class-independent 8-fold increase in circulating CD20
CD138
cells. No autoreactivity was found in this subset. These results argue against aberrant B cell development and rather suggest the autoantibody subclass predominance to be antigen-driven. The similarities between IgG4-AID suggest that, despite displaying variable clinical phenotypes, they share a similar underlying immune profile.
Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring ...due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings (11-color; 13 antibodies) and translational research (14-color; 16 antibodies). Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and
in vitro
functional assays, as well as for assessing the impact of sample preparation procedures and conditions. The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated (
vs.
manual “expert-based”) gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.
Summary
B‐cell precursors (BCP) regeneration in bone marrow (BM) after induction chemotherapy is prognostic for good treatment response in adult acute myeloid leukaemia (AML). We detected BCP ...regeneration in 81% of 59 paediatric AML patients at first complete remission; this compared to 46% in an adult study. BCP regeneration did not correlate with outcome or minimal residual disease levels. In 36 healthy BM controls, BCP levels were significantly higher in children as compared to adults. Therefore, BCP regeneration does not reflect good response to treatment in paediatric AML, possibly due to the relatively high base‐line levels of BCP in children.
Summary
A better understanding of the reconstitution of the B‐cell compartment during and after treatment in B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL) will help to assess the ...immunological status and needs of post‐treatment BCP‐ALL patients. Using 8‐colour flow cytometry and proliferation‐assays, we studied the composition and proliferation of both the B‐cell precursor (BCP) population in the bone marrow (BM) and mature B‐cell population in peripheral blood (PB) during and after BCP‐ALL therapy. We found a normal BCP differentiation pattern and a delayed formation of classical CD38dim‐naive mature B‐cells, natural effector B‐cells and memory B‐cells in patients after chemotherapy. This B‐cell differentiation/maturation pattern was strikingly similar to that during initial B‐cell development in healthy infants. Tissue‐resident plasma cells appeared to be partly protected from chemotherapy. Also, we found that the fast recovery of naive mature B‐cell numbers after chemotherapy was the result of increased de novo BCP generation, rather than enhanced B‐cell proliferation in BM or PB. These results indicate that post‐treatment BCP‐ALL patients will eventually re‐establish a B‐cell compartment with a composition and B‐cell receptor repertoire similar to that in healthy children. Additionally, the formation of a new memory B‐cell compartment suggests that revaccination might be beneficial after BCP‐ALL therapy.
Presence of malignant cells in cerebrospinal fluid (CSF) is a risk factor in pediatric acute lymphoblastic leukemia (ALL). Consequently, these patients receive extra intrathecal treatment. We ...evaluated the concordance between morphological and flow-cytometric (FCM) results, both on freshly analyzed and on stabilized, overnight transported samples.
Diagnostic CSF samples of 61 newly diagnosed pediatric ALL patients were divided in two aliquots. One was sent to the local laboratory and processed within a few hours after sampling (lab1). A second aliquot was 1:1 diluted with sterile medium and sent to the reference laboratory (lab2). For all samples, a MGG (May-Grünwald-Giemsa) stained cytocentrifuged slide was morphologically evaluated and two 6-color FCM stainings were performed. Samples were considered positive (CNS2) by MGG if at least one blast was seen and by immunophenotyping if a cluster (≥ 10 events) of ALL cells was detected.
Comparison of morphological data between both laboratories showed concordance in 33 samples (53%). In 20 of the 28 discordant samples only 1 or 2 blasts were reported by a single laboratory. Comparison of FCM data between laboratories was concordant in 58 samples (95%). Three samples (tumor load 15%-18%) were reported positive by one laboratory only; in two of these <100 cells could be acquired per tube. Comparison between FCM and morphology showed discordant results in 25 samples (41%) for lab1 and 19 (31%) in lab2. One discordant case was positive by FCM but negative by morphology, in all others only morphology was reported positive. The latter cases mostly had 1 or 2 blasts reported by morphology, generally not confirmed by the other laboratory.
This study shows that morphological analysis of CSF samples is non-reproducible if only 1 or 2 blasts are suspected. In contrast, FCM analysis of CSF is highly reproducible between fresh and stabilized samples. Although immunophenotyping still seems less sensitive than morphology, discordant samples generally have only 1 or 2 blasts reported by MGG, which is highly irreproducible. Nevertheless, by using a single 8-color tube higher cell numbers may be acquired and the FCM sensitivity may be increased.
No relevant conflicts of interest to declare.
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