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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. [ABSTRACT FROM AUTHOR]

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. 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). 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. 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." [ABSTRACT FROM AUTHOR]

Traditional manual gating strategies are often time-intensive, place a high burden on the analyzer, and are susceptible to bias between analyzers. Several automated gating methods have shown to exceed performance of manual gating for a limited number of cell subsets. However, many of the automated algorithms still require significant manual interventions or have yet to demonstrate their utility in large datasets. Therefore, we developed an approach that utilizes a previously published automated algorithm (OpenCyto framework) with a manually created hierarchically cell gating template implemented, along with a custom developed visualization software (FlowAnnotator) to rapidly and efficiently analyze immunophenotyping data in large population studies. This approach allows pre-defining populations that can be analyzed solely by automated analysis and incorporating manual refinement for smaller downstream populations. We validated this method with traditional manual gating strategies for 24 subsets of T cells, B cells, NK cells, monocytes and dendritic cells in 931 participants from the Health and Retirement Study (HRS). Our results show a high degree of correlation (r ≥ 0.80) for 18 (78%) of the 24 cell subsets. For the remaining subsets, the correlation was low (<0.80) primarily because of the low numbers of events recorded in these subsets. The mean difference in the absolute counts between the hybrid method and manual gating strategy of these cell subsets showed results that were very similar to the traditional manual gating method. We describe a practical method for standardization of immunophenotyping methods in large scale population studies that provides a rapid, accurate and reproducible alternative to labor intensive manual gating strategies. [ABSTRACT FROM AUTHOR]

Objective To elucidate the diversity of systemic lupus erythematosus (SLE) based on immunophenotyping. Methods Peripheral blood mononuclear cells were obtained from 143 SLE patients and 49 healthy individuals. Circulating B, T, and dendritic cells were defined using flow cytometric analysis as recommended by the Human Immunology Project Consortium. Based on these results, immunophenotypes were distinguished by principal components analysis (PCA), and cluster analysis was used to classify SLE patients into subgroups. Results The proportions of Treg and follicular helper T (Tfh) cells were higher in SLE patients than in healthy controls, whereas Th1 and Th17 cell proportions did not differ. Proportions of class-switched memory B cells and IgD-CD27- B cells were increased in SLE patients as well. The largest difference compared to the control group was observed in the proportion of plasmablasts, which was higher in SLE patients and correlated with disease activity as assessed with the British Isles Lupus Assessment Group index. PCA indicated that the immunophenotype of SLE patients consisted of abnormalities of the T and B cell axes. Cluster analysis showed that the SLE patients could be stratified into 3 subgroups (with high proportions of plasmablasts in all groups): patients who did not show the characteristic features (T cell-independent group), patients with a high percentage of Tfh cells (Tfh-dominant group), and patients with a high percentage of memory Treg cells (Treg-dominant group). The percentage of patients whose SLE was resistant to treatment was highest among the Tfh-dominant group. Conclusion Our study indicates that patients with active SLE can be divided into 3 subgroups based on T cell heterogeneity. Further immunophenotyping studies should help elucidate the pathogenesis of SLE and provide important information for the development of new therapies. [ABSTRACT FROM AUTHOR]

Introduction 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. [ABSTRACT FROM AUTHOR]

Objectives: To examine the utility of CD11c expression on monocytes in normal controls and patients with chronic myelomonocytic leukemia (CMML) (n = 23) with flow cytometric immunophenotyping. Methods: Twenty-three CMML samples and 10 control bone marrows submitted for lymphoma staging without evidence of disease were examined. Results: Monocytes in CMML samples ranged from 4% to 35%. Expression of at least one aberrant monocytic marker was found on the monocytes in 18 (82%) of 22 evaluable cases. The most common aberrancy was underexpression of CD11c (n = 15), while none of the bone marrow controls showed underexpression of CD11c. Conclusions: A distinct heterogeneous population of monocytic cells with underexpression of CD11c was identified in all these cases. CD11c underexpression was independent of other aberrancies, including HLA-DR underexpression (n = 14), aberrant CD56 expression (n = 11), and underexpression of CD33, CD38, and CD14 (n = 6, 5, and 5, respectively), supporting the utility of CD11c expression status on monocytes in establishing a CMML diagnosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Background: Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. Methods: We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. Results: Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ2 (PLCγ2), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. Conclusions: Genomic deletions in PLCG2 cause gain of PLCγ2 function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.) [ABSTRACT FROM PUBLISHER]

Objective: Acute mixed-lineage leukemia (AMLL) is characterized as the acute leukemia involved with acute myeloid cells and lymphoid cells at the same time. The AMLL is easily misdiagnosed because of a dual character involved with lymphoid and myeloid cells. At present, researches of AMLL in adults are more common. Only some are reported for children. Therefore, our aim was to study clinical characteristics of the childhood AMLL. Methods: From January 2000 to July 2009, 14 cases of AMLL children were selected by morphological and immunophenotyping methods from 185 cases of childhood acute leukemia admitted to the Department of Pediatrics, Tongji Hospital, Tongji Medical College of Science and Technology of Huazhong University. Medical records of all AMLL cases were reviewed for clinical characteristics. Findings: Fourteen cases of AMLL were screened from 185 cases of acute leukemia by morphology, immunology, cytogenetics and molecular (MICM). The rate of childhood AMLL accounted for 7.57% of pediatric acute leukemia (AL) diagnosed in the research period; white blood cell count in most of the patients was normal, the average value being 31.0×109/L in the first visit. In the 14 cases of AMLL, 8 cases were B-Ly+/My+, 2 cases were T-Ly+/My+B, and 4 were T+B-Ly+/My+. Among them, nine cases received treatment. Consequently, 6 cases reached complete remission (CR); 1 case had not complete remission; 2 cases did not complete the treatment. Conclusion: The diagnosis of AMLL should depend on the comprehensive evaluation of MICM. As there are still many problems concerning AMLL, it is very necessary that the research units collaborate with each other to improve the prognosis of childhood AMLL. The limitations and applications of the results are that they are only based on the patients of one hospital. [ABSTRACT FROM AUTHOR]

Abstract: 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 4.0×103 to 7.0×105 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The frequencies of human platelet-specific alloantigens (HPAs) vary between different ethnic groups, and genotyping using DNA techniques has been preferred over immunophenotyping methods for population studies. Using a polymerase chain reaction with allele-specific primers (PCR-ASP) method, we determined the allelic polymorphisms of five HPA systems among 174 unrelated individuals of two different Brazilian ethnic groups including Amazon Indians (n = 95) and blood donors (n = 79). Comparison of the calculated gene frequencies of the two alleles of HPA-1, -2, -3, -4 and -5 systems for Amazon Indians and Brazilian blood donors showed that gene frequencies obtained for the two alleles of HPA-1 (P < 0.001), HPA-2 (P = 0.001) and HPA-5 (P < 0.001) were significantly different between the two groups of individuals. All natives tested carried the HPA-2a and the HPA-5a alleles, but the HPA-1b and HPA-4b alleles are absent from the Indian population. It was also observed that all blood donors carried the HPA-1a, HPA-4a and HPA-5a alleles. In conclusion, the present data indicate differences in the frequency of the HPA systems between Amazon Indians and Brazilian subjects who present a high rate of racial admixture. While the frequencies of the HPA-1 and HPA-5 genes seen in Amazon Indians are similar to those reported for Oriental populations, the frequencies of the HPAs alleles in Brazilian blood donors are comparable to those reported for populations in North America and Europe. [ABSTRACT FROM AUTHOR]

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. 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". [ABSTRACT FROM AUTHOR]