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Helper innate lymphoid cells (ILCs), the most recently identified population of the ILC family, play a fundamental role in the restoration of tissue integrity, in the protection against infiltrating pathogens as well as in tumor immune-surveillance. ILCs have been divided into three main subsets, ILC1, ILC2, and ILC3, that can be specifically activated by different signals coming either indirectly from pathogens or from other cell populations, including cancer cells. Following activation, ILCs are in turn able to promptly secrete a wide range of soluble mediators that modulate effector cell functions. The discovery and the study of these immune cells is now offering important opportunities for innovative therapies of allergic airway diseases, inflammatory disorders and might be crucial for the discovery of new targets for the therapy of cancer. It is therefore fundamental that the scientific community establishes harmonized guidelines to obtain a consensus in the identification and phenotypical and functional characterization of ILCs. © 2018 International Clinical Cytometry Society.

International audience; Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease. Standardized procedures are essential to allow for inter-individual comparisons in the context of population based or clinical studies. Herein we report the approach taken by the Milieu Intérieur Consortium, highlighting the standardized and automated procedures used for immunophenotyping of human whole blood samples. We optimized eight-color antibody panels and procedures for staining and lysis of whole blood samples, and implemented pre-analytic steps with a semi-automated workflow using a robotic system. We report on four panels that were designed to enumerate and phenotype major immune cell populations (PMN, T, B, NK cells, monocytes and DC). This work establishes a foundation for defining reference values in healthy donors. Our approach provides robust protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.

Primary cutaneous gammadelta T-cell lymphoma (PCGD TCL), an aggressive type of lymphoma, accounts for approximately 1% of all primary cutaneous lymphomas. We have occasionally observed changes in T-cell antigen expression (immunophenotypic [IP] shift) over time, a phenomenon that is considered rare in T-cell lymphoma including cutaneous T-cell lymphoma. Therefore, we assessed sequential biopsies of PCGD TCL for possible IP shifts of the lymphoma cells. We searched for cases of PCGD TCL with consecutive biopsies to perform a comprehensive immunohistochemical analysis of paired specimens. A median of 12 markers per case was tested. We evaluated the percentage of neoplastic lymphocytes and determined the differential expression of antigens (gain, loss, increase or decrease). We identified 9 patients with PCGD TCL with consecutive biopsies. All (100%) cases had IP shifts of at least 1 antigen, whereas overall 22 pairs of markers were shifted: gain of reactivity occurred in 7 (31.8%) and loss in 3 (13.6%); increased reactivity in 4 (18.2%) and decreased in 8 (36.4%). Molecular analysis of TCRgamma showed identically sized monoclonal rearrangements between biopsy pairs in 4/4 (100%) patients. There was no correlation between IP shifts and the clinical appearance of lesions, histopathologic or cytologic features, or molecular rearrangements. IP shifts are common in PCGD TCL, occurring in all patients in this study and involving a variety of antigens. IP shifts do not seem to be linked to changes in the T-cell clone and are without obvious clinical or morphologic correlates. The occurrence of IP shifts in PCGD TCL suggests that antigen modulation may be involved in pathogenesis. IP shifts are somewhat frequent in T-cell lymphoma; however, it does not suggest a second neoplasm, and molecular studies can be used to determine clonal identity.

Background Human CD4+CD25+Foxp3+ T regulatory cells (huTreg) suppress CD4+ T cell-mediated antipig xenogeneic responses in vitro and might therefore be used to induce xenograft tolerance. The present study investigated the role of the adhesion molecules, their porcine ligands, and the chemoattractant factors that may promote the recruitment of huTreg to porcine aortic endothelial cells (PAEC) and their capacity to regulate antiporcine natural killer (NK) cell responses. Methods Interactions between ex vivo expanded huTreg and PAEC were studied by static chemotaxis assays and flow-based adhesion and transmigration assays. In addition, the suppressive function of huTreg on human antiporcine NK cell responses was analyzed. Results The TNFα-activated PAEC released factors that induce huTreg chemotaxis, partially inhibited by antihuman CXCR3 blocking antibodies. Coating of PAEC with human CCL17 significantly increased the transmigration of CCR4+ huTreg under physiological shear stress. Under static conditions, transendothelial Treg migration was inhibited by blocking integrin sub-units (CD18, CD49d) on huTreg, or their respective porcine ligands intercellular adhesion molecule 2 (CD102) and vascular cell adhesion molecule 1 (CD106). Finally, huTreg partially suppressed xenogeneic human NK cell adhesion, NK cytotoxicity and degranulation (CD107 expression) against PAEC; however, this inhibition was modest, and there was no significant change in the production of IFNγ. Conclusions Recruitment of huTreg to porcine endothelium depends on particular chemokine receptors (CXCR3, CCR4) and integrins (CD18 and CD49d) and was increased by CCL17 coating. These results will help to develop new strategies to enhance the recruitment of host huTreg to xenogeneic grafts to regulate cell-mediated xenograft rejection including NK cell responses.

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation., Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Primary cutaneous epidermotropic CD8-positive T-cell lymphoma represents an aggressive form of T-cell cutaneous lymphomas. Diagnosis is based on biopsies obtained from skin lesions. Here, we would like to report a case diagnosed by using flow cytometry performed on peripheral blood mononuclear cells. Moreover, an important finding was the difference in the results on targeting the CD8 antigen by using two different commercially available monoclonal antibodies. Perhaps, more than one antibody should be used in primary cutaneous aggressive epidermotropic CD8-positive T-cell lymphomas, in orderto be more accurate in the diagnosis and so in the classification of such diseases. © 2008 Clinical Cytometry Society

Background and objectiveFamilial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease (AID) worldwide. The disease is caused by mutations in the MEFV gene encoding the inflammasome sensor Pyrin. Clinical diagnosis of FMF is complicated by overlap in symptoms with other diseases, and interpretation of genetic testing is confounded by the lack of a clear genotype–phenotype association for most of the 340 reported MEFV variants. In this study, the authors designed a functional assay and evaluated its potential in supporting FMF diagnosis.MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from patients with Pyrin-associated autoinflammation with an FMF phenotype (n=43) or with autoinflammatory features not compatible with FMF (n=8), 10 asymptomatic carriers and 48 healthy donors. Sera were obtained from patients with distinct AIDs (n=10), and whole blood from a subset of patients and controls. The clinical, demographic, molecular genetic factors and other characteristics of the patient population were assessed for their impact on the diagnostic test read-out. Interleukin (IL)-1β and IL-18 levels were measured by Luminex assay.ResultsThe ex vivo colchicine assay may be performed on whole blood or PBMC. The functional assay robustly segregated patients with FMF from healthy controls and patients with related clinical disorders. The diagnostic test distinguished patients with classical FMF mutations (M694V, M694I, M680I, R761H) from patients with other MEFV mutations and variants (K695R, P369S, R202Q, E148Q) that are considered benign or of uncertain clinical significance.ConclusionThe ex vivo colchicine assay may support diagnosis of FMF and functional subtyping of Pyrin-associated autoinflammation.

Background aims Adoptive cellular therapy with immune effector cells (IECs) has shown promising efficacy against some neoplastic diseases as well as potential in immune regulation. Both inherent variability in starting material and variations in cell composition produced by the manufacturing process must be thoroughly evaluated with a validated method established to quantify viable lymphocyte subtypes. Currently, commercialized immunophenotyping methods determine cell viability with significant errors in thawed products since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte immunophenotypes in fresh and cryopreserved hematopoietic cellular products. Methods Using fresh or frozen cellular products and stabilized blood, we report on the validation parameters accuracy, uncertainty, precision, sensitivity, robustness and contamination between samples for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3–CD56+CD16+/– NK cells, CD19+ B cells and CD14+ monocytes of relevance to fresh and cryopreserved hematopoietic cellular products using the Cytomics FC500 cytometer (Beckman Coulter). Results The acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of ≤20% at three different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons) and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% coefficient of variation). Furthermore, the method can accurately report on the efficacy of cell depletion since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µL, respectively). The method complies with Foundation for the Accreditation of Cellular Therapy (FACT) standards for IEC, FACT-Joint Accreditation Committee of ISCT-EBMT (JACIE) hematopoietic cell therapy standards, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q2(R1) and International Organization for Standardization 15189 standards. Furthermore, it complies with Ligand Binding Assay Bioanalytical Focus Group/American Association of Pharmaceutical Scientists, International Council for Standardization of Hematology/International Clinical Cytometry Society and European Bioanalysis Forum recommendations for validating such methods. Conclusions The implications of this effort include standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection and in-process quality controls or dosing of IECs. This method also complies with all relevant standards, particularly FACT-JACIE standards, in terms of enumerating and reporting on the viability of the “clinically relevant cell populations.”

Introduction: Idiosyncratic DILI is a challenging condition, believed to involve the immune system. This hypothesis is supported by various identified HLA risk alleles. Objectives: To evaluate a potential role of the immune system in DILI through leukocyte immunophenotyping. Methods: Blood samples were collected from adjudicated DILI (n=12) and viral hepatitis (VH, 13) at day 1 (recognition), 7 and >30. A single blood sample was extracted from healthy liver controls (HLC, 54). Leukocyte populations and immune checkpoint expressions were determined based on cell surface receptors, except for CTLA-4 that was determined intracellularly, using multiparametric flow cytometry. Results: No differences were detected in leukocytes, lymphocytes or neutrophils counts at day 1. However, DILI (0.57 × 10E09/L, p=0.037) and HV (1.41 × 10E09/L, p

Investigating the dynamics of virus-host interactions in vivo remains an important challenge, often limited by the ability to directly identify virally-infected cells. Here, we combine detection of a beta-lactamase activated fluorescent substrate with full spectrum flow cytometry to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages by 8 days post-infection, and a concordant increase in small peritoneal macrophages and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC IIhigh large peritoneal macrophages emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 to 3 days post-infection, MHV68 infects ~5-10% of peritoneal cells, with >75% being large peritoneal macrophages. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in small peritoneal macrophages, with few infected dendritic cells and B cells. MHV68 infection at 3 days post-infection contains both lytic and latent infection, consistent with the identification of cells with active reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection.ImportanceIdentifying virally-infected cells in vivo is key to tracking viral infection and understanding host-pathogen interactions. The ability to further characterize and phenotype virally-infected cells is technically challenging. We use a mouse gammaherpesvirus, MHV68, expressing a reporter gene to identify infected cells during primary infection via flow cytometry. Optimization using this reporter system allowed us to further characterize infected cells via multiparameter full spectrum flow cytometry. Our study provides a technical model for high throughput single-cell immunophenotyping methods in the context of gammaherpesvirus infection. Furthermore, we show that acute MHV68 infection in the peritoneal cavity dramatically changes the immune landscape of this tissue, results in a high number of infected macrophages at early times, and is characterized by both lytic and latent infection within immune cells.

Purpose Biallelic pathogenic NBAS variants manifest as a multisystem disorder with heterogeneous clinical phenotypes such as recurrent acute liver failure, growth retardation, and susceptibility to infections. This study explores how NBAS-associated disease affects cells of the innate and adaptive immune system. Methods Clinical and laboratory parameters were combined with functional multi-parametric immunophenotyping methods in fifteen NBAS-deficient patients to discover possible alterations in their immune system. Results Our study revealed reduced absolute numbers of mature CD56dim natural killer (NK) cells. Notably, the residual NK cell population in NBAS-deficient patients exerted a lower potential for activation and degranulation in response to K562 target cells, suggesting an NK cell–intrinsic role for NBAS in the release of cytotoxic granules. NBAS-deficient NK cell activation and degranulation was normalized upon pre-activation by IL-2 in vitro, suggesting that functional impairment was reversible. In addition, we observed a reduced number of naïve B cells in the peripheral blood associated with hypogammaglobulinemia. Conclusion In summary, we demonstrate that pathogenic biallelic variants in NBAS are associated with dysfunctional NK cells as well as impaired adaptive humoral immunity.

Background: Flow cytometry is increasingly applied in cytopenic patients suspected for myelodysplastic syndromes (MDS). Analysis includes evaluation of antigen expression patterns in granulocytes of which, for example, partial lack of CD16 may indicate dysplasia, but presence of paroxysmal nocturnal hemoglobinuria (PNH)-type cells should be considered. However, diagnostic bone marrow (BM) samples hamper PNH analysis because immature stages in the granulo-/monocytic compartment lack expression of certain glycophosphatidyl-inositol-anchored proteins. In this prospective study, we evaluated the presence of PNH-type cells in BM next to aberrancies from routine MDS immunophenotyping. Methods: We combined antibodies defining maturation trajectories with FLAER. Validation of the designed method against routine PNH analysis and parallel analysis of BM and blood samples revealed similar results (granulocytes: Wilcoxon p = 0.25 and p = 0.82, respectively). We analyzed BM samples from 134 MDS, 17 chronic myelomonocytic leukemia, 15 aplastic anemia (AA), 1 PNH, 51 non-clonal cytopenic controls, and 12 normal controls. Results: Most AA/PNH-BM samples showed clear PNH clones: median 1.1% (0%–35%); CD16 loss on mature neutrophils paralleled PNH-clone sizes. In MDS-BM, only 3.7% of cases showed ≥0.1% PNH-type cells, whereas partial CD16 loss was more frequent and abundant. Conclusions: Our findings confirm that dysplastic features in MDS-BM may point to presence of PNH-type cells, though only few cases displayed FLAER-negative cells. We showed that identification of these cells in the granulocyte compartment of BM specimen is feasible, but—according to international guidelines—results need to be confirmed in peripheral blood.

Background Immune thrombocytopenia (ITP), autoimmune hemolytic anemia (AIHA) and autoimmune neutropenia (AIN) are disorders characterized by immune-mediated destruction of hematopoietic cell lineages. A link between pediatric immune cytopenias and inborn errors of immunity (IEI) was established in particular in the combined and chronic forms. Objective Identification of predictive factors of IEI in immune cytopenic children is an important objective for a prompt immunological diagnosis and appropriate management. Aim of this study is to detect clinical and laboratory signs of IEI, in particular the latter by an extensive lymphocyte immunophenotyping. Methods We retrospectively collected 47 pediatric patients with at least one hematological disorder among which persistent/chronic ITP, AIHA and AIN, aged 0–18 years at onset of immune cytopenias and/or immune-dysregulation. The cohort was divided into 2 groups (IEI+ and IEI-), based on the presence/absence of underlying IEI diagnosis. IEI+ group, formed by 19/47 individuals, included: Common variable immune deficiency (9/19), Autoimmune lymphoproliferative syndrome (4/19), DiGeorge syndrome (1/19) and unclassified IEI (5/19). Results IEI prevalence among patients with ITP, AIHA, AIN and Evans Syndrome was respectively of 42%, 64%, 36% and 62%. In IEI+ the extended lymphocyte immunophenotyping identified the presence of statistically significant (p-value

BackgroundHigh throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity amongst complex human cancers. scRNA-Seq studies using fresh human surgical tissue is logistically difficult, precludes histopathological triage of samples and limits the ability to perform batch processing. This hinderance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers, and on high throughput scRNA-Seq platforms.ResultsWe show that the viable cryopreservation of human cancers provides high quality single-cell transcriptomes using the Chromium 10X platform. We sequenced a total of ∼120,000 cells from fresh and cryopreserved replicates across three breast cancers, two prostate cancers and a cutaneous melanoma. Importantly, tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We then show that cryopreservation had minimal impacts on results of downstream analyses such as biological pathway enrichment. Further, we demonstrate the advantage of cryopreserving whole-cells for immunophenotyping methods such as CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing.ConclusionsOur study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

Background Tumor-associated microglia and macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) are potent immunosuppressors in the glioma tumor microenvironment (TME). Their infiltration is associated with tumor grade, progression, and therapy resistance. Specific tools for image-guided analysis of spatiotemporal changes in the immunosuppressive myeloid tumor compartments are missing. We aimed (i) to evaluate the role of fluorodeoxyglucose (18F)DPA-714* (translocator protein [TSPO]) PET-MRI in the assessment of the immunosuppressive TME in glioma patients, and (ii) to cross-correlate imaging findings with in-depth immunophenotyping. Methods To characterize the glioma TME, a mixed collective of 9 glioma patients underwent [18F]DPA-714-PET-MRI in addition to [18F]fluoro-ethyl-tyrosine (FET)-PET-MRI. Image-guided biopsy samples were immunophenotyped by multiparametric flow cytometry and immunohistochemistry. In vitro autoradiography was performed for image validation and assessment of tracer binding specificity. Results We found a strong relationship (r = 0.84, P = 0.009) between the [18F]DPA-714 uptake and the number and activation level of glioma-associated myeloid cells (GAMs). TSPO expression was mainly restricted to human leukocyte antigen D related–positive (HLA-DR+) activated GAMs, particularly to tumor-infiltrating HLA-DR+ MDSCs and TAMs. [18F]DPA-714–positive tissue volumes exceeded [18F]FET-positive volumes and showed a differential spatial distribution. Conclusion [18F]DPA-714-PET may be used to non-invasively image the glioma-associated immunosuppressive TME in vivo. This imaging paradigm may also help to characterize the heterogeneity of the glioma TME with respect to the degree of myeloid cell infiltration at various disease stages. [18F]DPA-714 may also facilitate the development of new image-guided therapies targeting the myeloid-derived TME.

SummaryIntroduction The workup of lymphoproliferative disorders (LPDs) involves the combined use of flow cytometry (FC) and immunohistochemistry (IHC). This often results in duplicate immunophenotypic testing and adds costs that may not be eligible for reimbursement based on the Medicare National Correct Coding Initiative. We aimed to establish a cost-effective diagnostic algorithm based on initial FC categorization to reduce repetitive immunophenotyping. Methods We retrospectively reviewed 242 cases of suspected LPDs with concurrent FC and IHC testing over a 12-month period. We correlated FC with surgical diagnoses and evaluated the frequency of repeat IHC testing. Results Repetitive immunophenotyping was common; overall, 85% of cases had at least one marker repeated. Concordant cases were significantly less likely to have markers repeated than discordant cases. Of concordant B cell malignancies, 57% represented recurrent disease; however, repeat marker usage was not decreased as compared to new diagnoses. The most frequently repeated markers were CD3, CD5, CD10, and CD20. Conclusions We propose that in concordant cases, CD5 and CD10 should not be repeated by IHC; this would decrease the use of these markers by 80% and 76%, respectively. We developed an algorithmic approach to IHC usage that has improved incorporation of FC data at our institution and may reduce healthcare costs.

A key event in cancer progression is the establishment of an immunosuppressive environment that prevents the immune system from effectively attacking the cancer cells. In this study we aim to characterize the cellular determinants and the spatial features of immune suppressive mechanisms in high-grade serous ovarian cancer (HGSOC) via integrating tissue Cyclic Immunofluorescence (tCycIF), a novel highly multiplexed single-cell spatial proteomics platform with multi-omics data, including state-of-the-art single-cell RNA sequencing. We have conducted a comprehensive analysis of a unique dataset consisting of 23 HGSOC samples, including 7 pairs of treatment-naïve and post-chemotherapy samples, using tCycIF, single-cell, and bulk RNA sequencing coupled with novel deconvolution algorithms and whole-genome sequencing. All samples were prospectively collected from patients treated at Turku University Hospital, Finland, as a part of the HERCULES consortium. Our preliminary findings suggest that tCycIF can reliably capture tumor microenvironment composition in HGSOC. Overall, there was a good correlation with tumor purity between all the methods used. In tCycIF, scRNAseq and bulk deconvolution macrophages were the most common immune cell type in the HGSOC microenvironment. However, specific immune subpopulations, such as CD8+T-cells, had a larger proportion in tCycIF compared to single-cell RNA sequencing and bulk-deconvoluted RNAseq data. Further, the immune profiling based on RNAseq deconvolution algorithm showed highly variable results based on the algorithm used. We are in the process of using our complete dataset to compare state-of-the-art methods and improve algorithms for HGSOC immunophenotyping. tCycIF’s spatial proteomic data in single-cell resolution allows us to gain new insights into the tumor microenvironment and discover the previously latent cell-to-cell interactions that make up the immunosuppressive milieus in HGSOC tumors. The improvement of novel reliable single-cell immunophenotyping methods is critical to develop more effective immunotherapies to improve the outcomes and survival of patients with HGSOC. Citation Format: Miikka K. Kilkkila, Julia Casado, Connor Jacobson, Antti Hakkinen, Erdogan Erkai, Jun Dai, Kaiyang Zhan, Zoltan Maliga, Olli Carpen, Rainer Lehtonen, Sampsa Hautaniemi, Peter Sorger, Anna Vaharautio, Anniina Farkkila. Integrating highly-multiplexed imaging with multi-omics data to uncover immunologic vulnerabilities in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B62.

Background & Aim Adoptive cellular therapy with immune effector cells (IEC) has shown remarkable toxicity against tumor cells and potential in immune regulation. Both variability in cell content of starting material and compromised cell viability during manufacturing necessitate a validated method for quantifying viable lymphocyte subtypes individually. Currently-commercialized immunophenotyping methods report cell viability in thawed products with significant error since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte- immunophenotypes in fresh and cryopreserved hematopoietic cellular products. Methods, Results & Conclusion Using fresh or frozen cellular products and stabilized blood, we report on the validation parameters: accuracy, uncertainty, precision, sensitivity, robustness and carry-over for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3-CD56+CD16+/- NK cells, CD19+ B cells and CD14+ monocytes of relevance to hematopoietic products and immune effector cell (IEC) products on the Cytomics FC500 Beckman Coulter cytometer. The acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of 10% at 3 different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons), and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% CV). Furthermore, the method can accurately report on the efficacy of cell depletion, since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µl, respectively). The method complies with FACT Standards for IEC, FACT-JACIE Hematopoietic Cell Therapy Standards, ICH Q2(R1) and ISO15189 standards. Furthermore, it complies with LBAFG-AAPS, ICSH/ICCS and European Bioanalysis Forum recommendations for validating such methods. The implications of this effort include: standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection, in-process or dosing of immune effector cells. This method also complies with all relevant standards, particularly FACT-JACIE Standards in terms of enumerating and reporting on the viability of the “clinically relevant cell populations”.

Introduction: Immunosuppression of T lymphocytes is required for preventing acute rejection after transplantation and for the treatment of chronic autoimmune and inflammatory diseases. The laboratory monitoring for this therapy is the measurement of T cells by immunophenotyping, aiming the target value of less than 20 cells per µL. Objective: To establish a cut-off point for the total number of lymphocytes in the automated blood cell count that reflects less than twenty T cells µL by immunophenotyping. Methods: We studied and evaluated 242 kidney transplant patients that had results of automated blood cell count and quantification of T cells by immunophenotyping technique. The patients were divided into two groups, depending on the T lymphocyte immunophenotyping rates established by lower and higher than 20 cells per µL. After, we evaluated the cut-off point for lymphocytes in the blood cell count with a specificity of 100% to exclude patients with high levels of T lymphocytes. Results: We found that the cut-off point of 70 lymphocytes per µL obtained by automated blood cell count showed 100% of specificity to exclude patients with T-cell counts higher than 20 cells per µL by immunophenotyping. Conclusion: The results found in this study may be helpful to monitor the immunosuppressive therapy in kidney transplant patients in places where a flow cytometer is not available, or when this equipment is not present in the full routine. Resumo Introdução: A imunossupressão de linfócitos T é necessária para a prevenção da rejeição aguda em transplantes e no tratamento de doenças autoimunes e inflamatórias crônicas. O seu monitoramento laboratorial consiste na quantificação dos linfócitos T realizada pela técnica de imunofenotipagem, na qual o valor preconizado é manter inferior a 20 células/µL. Objetivo: Estabelecer um ponto de corte para o número de linfócitos totais no hemograma automatizado que reflita uma contagem de linfócitos T inferior a 20 células/µL por imunofenotipagem. Métodos: Foram avaliados 242 pacientes transplantados renais que continham resultados do hemograma automatizado e quantificação de linfócitos T por imunofenotipagem. Os pacientes foram divididos em dois grupos, conforme os valores de linfócitos T estabelecidos pela imunofenotipagem: inferiores e superiores a 20 células/µL. A partir disto, foi avaliado o ponto de corte de linfócitos no hemograma com especificidade de 100% para excluir os pacientes com valores elevados de linfócitos T. Resultados: Este estudo evidenciou que o ponto de corte de 70 linfócitos/µL obtidos pelo hemograma automatizado apresentou especificidade de 100% para excluir os pacientes com contagens de linfócitos T superiores a 20 células/µL na imunofenotipagem. Conclusão: Esta pesquisa poderá auxiliar no monitoramento da terapia imunossupressora em pacientes transplantados renais em locais que não possuem um citômetro de fluxo disponível, ou ainda quando este equipamento não se faz presente na rotina integral.

Background and aims To elucidate the diversity of systemic lupus erythematosus (SLE), we stratified SLE patients based on immunophenotyping. Methods Peripheral blood mononuclear cells were obtained from 80 active SLE patients (with one or more BILAG category A, or two or more BILAG category B). Circulating B, T and dendritic cells were defined based on flow cytometric analysis for human immune system termed ”the Human Immunology Project”. Based on these results, the immunophenotype was visualised by principal component analysis and SLE patients classified into subgroups by cluster analysis. Results Principal component analysis indicated that the immunophenotype of active SLE patients was consistent with T and B cell axes. Among these correlations, Th17 and Treg cells were statistically close, and showed positive correlation (p Conclusions Our study indicates that SLE patients can be divided into three subgroups based on T cell heterogeneity. This heterogeneity should be taken into consideration not only in basic research but also in patient selection in clinical trials for development of new drugs.

Malignant lymphomas are a group of cancerous diseases that develop from lymphocytes and primarily affect lymph nodes. Being the sixth most common cancer type in Sweden, lymphoma is a societal problem that needs to be tackled by improving care and treatment of patients. This study was designed to examine the blood cell composition in lymphoma patients and well as determine whether the use of cryopreserved cells affected the analysis outcome. An evaluation of the methods used was also performed. Frozen peripheral blood from lymphoma patients as well as fresh and frozen blood from healthy controls was used. The cells of interest were monocytes, granulocytes, Treg, NKT, iNKT, B and T cells plus the dendritic cell activation protein CCR7. Three immunophenotyping methods were used. Method one was used in staining surface cell markers while the other two were for both surface and intracellular staining using two distinctive kits. The results showed no significant difference in immune cell composition between patients and blood donors. Limited patient samples and the lack of female blood donors could explain the unexpected result. A substantial difference in Treg cells was observed in fresh and frozen tested samples as well as T cell outcomes in method one compared to the other two methods. There were fewer Treg cells in frozen samples, which probably was due to cryopreservation while the lack of fixation in method one led to the loss of CD4+ T cells. Overall, the methods used were adequate but definitely require some improvements.

Objectives: To assess a large series of patients with acute myeloid leukemia (AML) with t(8;21) for both IGH@ and IGK@ B-cell gene rearrangements and for expression of PAX5, OCT2, and Bob.1 by immunohistochemistry and expression of CD19, CD79a, CD20, and CD22 by flow cytometry immunophenotyping. Methods: A total of 48 cases of AML with t(8;21)(q22;q22) were evaluated by immunohistochemistry and/or heavy chain and light chain immunoglobulin rearrangement studies where paraffin-embedded and/or fresh frozen material was available for study; previously performed flow cytometry studies were also reviewed in available cases. Results: Our study yielded 1 of 19 cases of AML with t(8;21) with an IGH@ gene rearrangement; blasts were associated with weak PAX5 expression. In addition, expression of antigens CD79a by flow cytometry and OCT2 by immunohistochemistry were highly associated with PAX5 expression, and CD19 was expressed in most cases assessed. Conclusions: Although B-cell antigen and B-cell transcription factor expression is seen in the majority of AMLs with t(8;21)(q22;q22) and correlates with PAX5 expression, immunoglobulin gene rearrangements are an uncommon event in this group of leukemias.

Objective Intraocular lymphoma (IOL) is a rare condition and frequently difficult to distinguish from uveitis or other uveitis-masquerading syndromes. The diagnosis is confirmed by cytologic examination of ocular fluid specimens and more recently by molecular-immunoglobulin heavy chain (IGH) translocation or cytokine analysis. However, some of these more recent methods have not been validated by follow-up studies. Design Evaluation of a diagnostic test. Participants In a cohort of 51 consecutive patients with a clinical suspicion of IOL, vitreous analysis was performed via multicolor flowcytometric immunophenotyping. Methods Multicolor flowcytometric immunophenotyping was performed with CD45, CD3, CD19, CD20, anti-SmIgκ, and anti-SmIgλ antibodies. The presence of a clear B-cell population showing a disequilibrium of Igκ versus Igλ expression was used to confirm the diagnosis of non-Hodgkin lymphoma (NHL). Patients were followed for a minimum of 2 years (mean, 5.9±2.0 years) to validate the accuracy of the method. Main Outcome Measures The presence or absence of IOL during follow-up. Results In 14 of 51 patients, a clinical diagnosis of IOL was confirmed using flowcytometric analysis. Of these 14 patients, 11 had primary IOL and 3 had metastasized secondary lymphomas. In 3 of 51 patients who were diagnosed with (central nervous system) NHL during follow-up, the test failed to confirm the presence of a clonal B-cell population. In 18 of the 34 other patients, an infectious or well-defined immunologic disorder was established during follow-up. The remaining 16 patients, with a minimal follow-up of 2 years, were diagnosed with idiopathic uveitis. Conclusions Multicolor flowcytometric analysis had 82.4% sensitivity and 100% specificity in patients with suspected IOL. This is comparable to the reported vitreous interleukin (IL)-6/IL-10 testing sensitivity of 0.8 and sensitivity of 0.65 to 0.95 by immunoglobulin heavy chain (IGH) gene arrangement testing in clinical cohorts. Because flowcytometric tests are readily performed in hematologic laboratories, this can be regarded as a useful method for confirming the clinical diagnosis of IOL. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Based on the aptamer-conjugated core-shell near-infrared fluorescent nanoparticles (NIR-Nps) and fluorescence anisotropy measurement, the present study reported proof-of-principle for a rapid homogeneous assay approach that can detect target cancer cells without the need of the complicated separation steps in whole blood samples. Experimental investigation showed that the novel NIR-Nps have negligible background fluorescence and low inner filtration interference in complex biologic systems such as whole blood. The specific recognition characteristic of aptamer in whole blood samples was investigated by using the proposed fluorescence anisotropy method. The results showed that the fluorescent nanoparticle-tagged aptamer probes sequence could achieve specific recognition of the target cancer cells from complex mixtures including whole blood samples. And the reaction conditions for the binding between fluorescent nanoparticle-conjugated aptamer probes and target cancer cells were optimized. The present approach can exhibit sensitive and reproducible fluorescence anisotropy responses to the target cells concentration and the calibration curve showed good linearity when the target cells concentration is in the range from 4.0 x 10(3) to 7.0 x 10(5)cells/mL. Moreover, the present fluorescence anisotropy assay technique could be practically utilized for the detection of acute leukemia samples with improved capabilities and be comparable to the immunophenotyping methods clinically used.

Aims: Tissue microarrays offer an efficient way of examining a large number of tumour cases on a single glass slide. A major concern, however, is tumour heterogeneity. Also, the use of tissue microarrays in biopsy material is unexplored. The purpose of the present study was to assess the possibility and validity of arraying three 0.6-mm cores per case in endoscopic gastric cancer biopsies for immunophenotyping. Methods and results: Thirty-eight cases were studied with immunohistochemical staining for p53, CD44v6 and vascular endothelial growth factor. Full tissue sections were compared with triple core-tissue microarrays. Thirty-six cases contained three cores with tumour, one case contained two cores with tumour and one case contained only a single core with viable tumour and was excluded. Three further cores had been lost from three separate cases on the sections for immunohistochemistry. κ values for whole-sections versus tissue microarrays ranged between 0.77 and 0.94. p53 immunohistochemical staining (interpretation as + or –) yielded the best result with only 1/37 mismatches, whereas CD44v6 (graded both for intensity and extent) showed 3/37 mismatches. The small depth of tissue in cores from biopsies necessitates all cores being arrayed flush with the face of the recipient wax block for maximizing the number of sections available. Compared with the first section over 30 additional 4-µm sections were available before the first case (with one core left) had to be excluded and 80 sections before half the tissue cores were lost. Conclusions: It is impracticable to array more than 120–150 cores per block. Tissue microarray with three cores per case is feasible and valid for studying biopsy material.