Immunophenotyping Panel Research Articles

Defining the Cell Surface Landscape in B-Cell Malignancies: Comprehensive Profiling of Surface Antigens for Biomarker Discovery and Patient Stratification

Flow cytometric analysis of surface marker expression is a fundamental component of diagnosis, classification, and monitoring for many hematologic malignancies. However, most clinical implementations of surface immunophenotyping focus on only a handful of markers. A comprehensive survey of CD markers and cell-surface receptors could provide a higher resolution and more accurate characterization of cancer lineages, as well as enabling new insights into the cellular changes resulting from disease progression or therapeutic intervention. To this end, we collated and evaluated antibodies to over 400 known markers and receptors. To establish the baseline and variability of each marker in major B cell subsets within normal human blood, a protocol was established to co-stain each sample for CD19, CD20, CD27, IgD, CD38, and CD24 simultaneously with each of the 400 markers. Antigen abundance and expression frequency were evaluated to establish a surface marker profile for each B-cell subset and to establish the variation inherent in each marker across the normal human population. To validate the application of this panel for cancer immunophenotyping, 20 malignant B-cell lines were selected for comprehensive surface characterization. Prior to staining, each cell line was barcoded with a combination of fluorescent dyes to generate a unique fluorescent signature. This approach allowed multiple cell lines to be combined into a single well for staining and analysis, enabling the rapid evaluation of hundreds of surface antigens using only 1-2 million cells from each line. Hierarchical clustering of data from cancer lines and normal B cell subsets allowed the identification of markers useful for discriminating between B-cell cancer subtypes, as well as markers that differed between normal and malignant B cells. These studies have resulted in the optimization of a protocol and antibody panel for use in profiling surface protein expression on human blood cells and cell lines. Application of this procedure to clinical samples may provide information useful for biomarker discovery, improved stratification of hematologic malignancies, and the identification of new drug targets. DisclosuresO’Donnell:Primity Bio: Employment. Alvendia:Primity Bio: Employment. Wehrman:Primity Bio: Employment. Krutzik:Primity Bio: Employment.

Minimal Residual Disease Sensitivity in Patients with Pre-B Cell Acute Lymphoblastic Leukemia Using Gemstone Analysis Method

The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and to avoid over- or under- treatment. Minimal residual disease (MRD) is a very effective method for predicting response to treatment and potential relapse in patients with ALL. The most validated method used to assess MRD in ALL is flow cytometric (FCM) analysis of leukemia-associated immunophenotypes which can detect up to 1 leukemic cell per 10,000 normal cells (0.01%). There is no standard method for detecting MRD by FCM in ALL patients. Our institutional approach has been to evaluate 20,000- 50,000 events using a specific immunophenotypic panel and to analyze the data using WinList software with a concurrent comparison with the diagnostic immunophenotype.GemStone™ is a new data analysis program that uses a Probability State Modeling (PSM) approach to analyze FCM data. The incorporated TriCOM technology numerically correlates all the immunophenotype combinations which can be used to detect MRD and other rare populations.In this study, we compared the GemStone and WinList analysis methods on the same patient data files. We identified 33 patients suitable for comparison by performing a retrospective review of the medical records from 2004-2010 in our pre-B cell ALL database. Patients were newly diagnosed, untreated, age ≥17 years and had FCM data available at diagnosis, week 4 (range 3-8) and week 16 (range 12-20) post therapy. We analyzed the FCM files to detect MRD using both GemStone and WinList methods separately, we subsequently reviewed the clinical course of the patients and identified those who sustained complete remission or relapsed. The clinical and FCM results were statistically correlated in Chi Square 2x2 table to identify the predictive value of each analysis method.WinList: we used CD10, CD19, CD34 and CD38 as a backbone marker set and tracked the same abnormal immunophenotype upon diagnosis in the subsequent follow up samples.GemStone: We used the same marker set and incorporate the TriCOM technology identify the MRD numerically.Figure 1 compares the outcome between GemStone and WinList methods at 16 weeks follow up.Table 1 reviews Chi Square with both method of analysis which gives comparable results.In summary, our retrospective review showed that GemStone method in detecting the MRD had comparable clinical and statistical significance to the WinList method. However, we found that the limited number of events collected (20,000) reduced our ability to completely automate the analysis process in GemStone due to intensity differences in the data. We are now conducting a prospective analysis by GemStone on ALL patients with higher number of collected events (5 x 10⁵- 2x 10⁶)Further, in our review, the MRD at 4 months post diagnosis (average 12-20 weeks) was more clinically significant than the one at 4 weeks follow up (range 3-8 weeks). [Display omitted] Abstract 5348Table 1: comparison between WinList and GemStone method.SensitivitySpecificityPPVNPVP valueStatistically significantWinList at 4 week F/U33%58%23%70%0.49noGemStone at 4 week F/U44%79%44%79%0.17noWinList at 16 week F/U78%58%41%88%0.0323yesGemStone at 16 week F/U89%67%50%94%0.0022yes DisclosuresHill:Verity Software house: Employment. Griffiths:Astex Pharmaceuticals: Research Funding; Celgene, Incyte and Alexion: Honoraria.

Abstract 2496: Defining the cell surface landscape in cancer: Comprehensive profiling of surface antigens for biomarker discovery and immune monitoring

Abstract Flow cytometric analysis of surface marker expression is a fundamental component of diagnosis, classification, and monitoring for many hematologic malignancies. However, most clinical implementations of surface immunophenotyping focus on only a handful of markers. A comprehensive survey of CD markers and cell-surface receptors could provide a higher resolution and more accurate characterization of cancer lineages, as well as a better understanding of the immune response to disease progression or therapeutic intervention. To this end, we collated and validated 400 antibodies to known markers and receptors. Antibodies were validated for their performance on live and formaldehyde-stabilized samples to ensure compatibility with standard processing procedures. To establish the baseline and variability of each marker in the normal human population, a protocol was established to co-stain each sample for CD3, CD19, CD16, CD56, and CD14 simultaneously with each of the 400 markers. Differences in antigen abundance and expression frequency were calculated to establish the variation inherent in each marker across the population. To further validate the application of this panel for cancer immunophenotyping, eight commonly studied leukemia and lymphoma cell lines were selected for comprehensive surface characterization. Prior to staining, each cell line was fixed and barcoded with a combination of fluorescent dyes to generate a unique fluorescent signature. This approach allowed multiple cell lines to be combined into a single well for staining and analysis, enabling the rapid evaluation of hundreds of surface antigens using less than five million cells from each line. These studies have resulted in the optimization of a protocol and antibody panel for use in profiling surface protein expression on fixed human blood cells and cell lines. Application of this procedure to clinical samples may provide information useful for biomarker discovery, improved stratification of hematologic malignancies, and the identification of new drug targets. Citation Format: Erika A. O'Donnell, Erica L. Alvendia, Benjamin E. Osetek, Tom S. Wehrman, Peter O. Krutzik. Defining the cell surface landscape in cancer: Comprehensive profiling of surface antigens for biomarker discovery and immune monitoring. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2496. doi:10.1158/1538-7445.AM2014-2496

Vaccination with Irradiated Autologous Tumor Cells Mixed with Irradiated GM-K562 Cells Provokes Lymphocyte Activation and Specific Antitumor Immunity in Patients with Recurrent Glioblastoma

We have previously reported the safety and feasibility of vaccinating recurrent malignant glioma patients with irradiated autologous cells mixed with irradiated GM-K562 cells. Here, we report the impact of vaccination on the expression of T-lymphocyte co-stimulatory and co-inbhibitory molecules as well as on regulatory T-lymphocytes. Furthermore, we explored the impact of vaccination on patient humoral immune responses with particular attention to antibody titers against molecules associated with tumor angiogenesis. Blood was collected from 9 of 10 treated patients at regular intervals and submitted for immune analysis. 5-color flow cytometry was employed after staining of whole blood by an immunophenotyping antibody panel. Antibody titers to tumor-associated antigens were measured by ELISA of patient plasma - all assays were performed in duplicate. Treatment-associated change in percent expression of various markers was analyzed via the non-parametric sign-rank test. p < 0.05 was considered statistically significant. In patients undergoing craniotomy for recurrent malignant glioma, vaccination with irradiated autologous tumor cells and irradiated GM-K562 cells was associated with statistically significant increases in CD4+ T-lymphocyte expression of costimulatory molecules OX40 and 4-1BB (CD137). CD8+ T-lymphocytes saw increased expression of 4-1BB as well. Likewise, there were increases in CD4+ T-lymphocyte expression of CTLA-4, PD1, and FOXP3 - each of which is associated with negative immune regulation and are targetable with existing monoclonal antibodies. 7 of 9 patients had marked treatment-driven increases in antibody titers to Angiopoietin 2, which may represent a novel glioma vasculature-associated antigen. Vaccination with irradiated autologous tumor cells mixed with irradiated GM-K562 cells in patients with recurrent malignant glioma is biologically active and activates both T-lymphoctes and humoral antitumor immunity. Vaccine-induced expression of costimulatory molecules may provide the rationale for combination therapies with either co-stimulatory receptor agonist or co-inhibitory receptor antagonist molecules.

First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian group of flow cytometry-GBCFLUX

Multiparameter flow cytometry is a highly sensitive, fast, and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable, and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an interlaboratory study to evaluate its effectiveness on the diagnosis and classification of AL. (Assoc editor comm. 2).

"First proposed panels on acute leukemia for four-color immunophenotyping by flow cytometry from the Brazilian Group of Flow Cytometry - GBCFLUX"

Multiparameter flow cytometry (MFC) is a highly sensitive, fast and specific diagnostic technology with a wide range of applicability in hematology. Although well-established eight-color immunophenotyping panels are already available, most Brazilian clinical laboratories are equipped with four-color flow cytometer facilities. Based on this fact, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) for standardization of clinical flow cytometry has proposed an antibody panel designed to allow precise diagnosis and characterization of acute leukemia (AL) within resource-restricted areas. Morphological analysis of bone marrow smears, together with the screening panel, is mandatory for the primary identification of AL. The disease-oriented panels proposed here are divided into three levels of recommendations (mandatory, recommendable and optional) in order to provide an accurate final diagnosis, as well as allow some degree of flexibility based on available local resources and patient-specific needs. The proposed panels will be subsequently validated in an inter-laboratory study to evaluate its effectiveness on the diagnosis and classification of AL. © 2014 Clinical Cytometry Society.

Erythroid Lineage Cells Are Found In Close Association With Bone In The Marrow Microenvironment

Osteolineage cells within the bone marrow microenvironment have been implicated in support and regulation of hematopoietic stem cells (HSCs). Recently, augmented hypoxia-inducible factor (HIF) signaling in osteoprogenitors has been shown to expand the HSC niche, and surprisingly these cells have also been demonstrated to express erythropoietin, the critical cytokine stimulating erythropoiesis. We therefore hypothesize that endosteal cells may represent an additional regulatory site for erythropoiesis. To further delineate the role of the osteolineage cells in the support of erythropoiesis, we isolated bone associated cells (BACs) with enzymatic digestion of adult C57bl/6 mice hind limbs after bone marrow flushing and depleted the BACs of CD45+ cells to enrich for osteogenic cells.We suspected some contribution of erythroid cells to CD45- BACs, however we were surprised to find that ter119+ cells represented a large percentage of BACs after enzymatic digestion. After CD45 depletion, ter119+ cells constituted about 30% percent compared to approximately 0.85% of CD45+ cells (33 ± 4.4vs. 0.85 ± 0.26, p= 0.0018) by flow cytometric analysis. Additionally, CD45 depleted BACs had approximately 46 fold higher osteocalcin expression than CD45+ cells (1300 ± 120 vs. 28 ± 9.5, p < 0.0001), while CD45/Ter119/CD31 depleted BACs had approximately 2000 fold higher osteocalcin expression than CD45/Ter119/CD31 (+) cells (2000 ± 520 vs. 0.98 ± 0.02, p= 0.0044) by qRT-PCR, confirming enrichment of the osteoblastic lineage by this immunophenotypic panel. These data suggest that there are a large number of erythroid lineage cells associated with the BACs along the endosteum. In the bone marrow of adult mice, ter119 + cells represented approximately 85% in the CD45- pool as compared to 5% in the CD45+ cell pool.To determine if the endosteum is an active site of erythropoiesis, we quantified erythroid progenitors and precursors in the BAC pool compared to whole bone marrow (wbm) and peripheral blood (pb) by both flow cytometric analysis and colony forming assays. Flow cytometric analysis demonstrated the presence of every phase of erythroid differentiation in the BAC pool, including the presence of phenotypic MEPs (wbm vs bac vs pb: 250 ± 30 vs 84 ± 22 vs 0), BFU-E (wbm vs bac vs pb: 300 ± 14 vs 110 ± 36 vs 0 ), CFU-E (wbm vs bac vs pb: 2900 ± 2 vs 430 ± 23 vs 1 ± 0.8) and proerythroblasts (wbm vs bac vs pb: 11000 ± 2500 vs 7600 ± 1600 vs 2300 ± 920) per million cells. The phenotypic frequency of CFU-E was particularly remarkable in the BACs (430 ± 23) as compared to peripheral blood (1 ± 0.8) , demonstrating that all stages of erythroid differentiation are found in tight association with the endosteum and are not due to contamination from circulating erythroid progenitors. Colony assays were performed for CFU-E (wbm vs. bac 108 ± 16 vs 6.3 ± 2 colonies per 20,000cells plated), BFU-E (wbm vs. bac 55 ±1.0 vs 2 ±1.0; colonies per 40,000 cells plated) and myeloid progenitors (wbm vs. bac 66 ± 28 vs 11 ± 2.5 ; colonies per 10,000 cells plated) also confirmed the presence of erythroid progenitors at endosteal sites.Together these results identify the endosteal surface as a site for erythroid differentiation. The presence of all phases of erythroid lineage differentiation in the BACs suggests a potential role for osteolineage cells for maintenance and regulation of erythropoiesis. Whether osteolineage cells contribute to erythroid lineage homeostasis and/or stress response, and whether activation or damage to osteolineage cells alters local erythroid differentiation remains to be demonstrated. However our data suggest further study of the endosteum and osteolineage cells as a potential and unexpected site of erythroid regulation, which could potentially be targeted to accelerate erythropoiesis and treat anemia. Disclosures:No relevant conflicts of interest to declare.

Analysis Of Immune Cell Populations Before and After Autologous Hematopoietic Stem Cell Transplant For Multiple Myeloma: Association With Early Recovery Of Absolute Lymphocyte Count and Progression-Free Survival

The treatment of transplant-eligible, multiple myeloma (MM) patients usually consists of induction therapy followed by autologous hematopoietic stem cell transplant (ASCT). In addition to typical cytogenetic and biochemical prognostic factors, there are other risk factors that predict for relapse and progression after ASCT. This includes absolute lymphocyte count (ALC) recovery (>0.5 cells/μL) by day +15 post ASCT, which has previously been shown to correlate with prolonged progression-free (PFS) and overall survival (OS). In a cohort of 70 MM patients who received high dose melphalan with ASCT between 8/2007 and 9/2012, we performed a comprehensive immunophenotyping panel including 35 T-, B-, NK- and Dendritic Cell (DC)-cell subsets a median of 25 days before ASCT (N=70), at day +30 post-ASCT (N=40) and at day +100 (N=51) post-ASCT. Specifically, the immunophenotyping panel included total numbers of T cells (CD3+, CD4+, CD8+), B cells (CD19+, CD20+), NK cells (CD56+CD16+) and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells. T cell subsets included the relative proportions of double positive (CD3+CD4+CD8+), double negative (CD3+CD4-CD8-), CD4+ and CD8+ Naïve (CD3+ CD45RA+ CD45RO- CD27+), CD4+ and CD8+ central memory (CD3+ CD45RA- CD45RO+ CD27+), CD4+ and CD8+ effector memory (CD3+ CD45RA- CD45RO+ CD27-), recent thymic emigrants (CD3+ CD4+ CD45RA+ CD31+), and T-regulatory cells (CD3+ CD4+ CD25(br), CD3+CD4+CD25+CD127(d), CD3+CD4+CD25+CD127-HLADr+). B cell subsets included proportions of naïve (CD19+ CD27-), and memory (CD19+ CD27+, pre-switch CD19+ IgD+ CD27+, post-switch CD19+ IgD- CD27) cells. All patients survived to day +100. We examined MM response to ASCT, recovery of ALC by day +15 post-ASCT, clinically significant infections, PFS and OS. Characteristics in 70 patients pre-ASCT were: median (range) age 60 (28-75) yrs; 54% females; 26% KPS ≥90; response to most recent therapy was 13% in CR/sCR, 40% in VGPR, 41% in PR, 6% stable disease. MM responses at day + 100 included: 51% achieved or maintained CR/sCR, 17% VGPR, 10% PR, 24% SD and 6% progressed. The frequency of different immune cell subsets at either pre-ASCT or day +100 post-ASCT were not associated with CR/sCR status at day 100 post-AHSCT. ALC recovery by day +15 post-ASCT was associated with significantly longer median PFS (651 vs 288 days, P=0.001) and OS (804 vs 391 days, P=0.02) than those who did not recover ALC by day 15. For patients showing early recovery of ALC, the absolute numbers of T-cells pre-ASCT (P=0.08), as well as the proportion of CD4+ naïve (P=0.07) and CD8+ NK (CD8+/CD16+/CD56+, P=0.06) populations tended to be higher, whereas the absolute counts of gamma-delta T cells (P=0.02), CD4+ helper (P=0.03, See Figure), CD8+ effector (P=0.02) T-cells and total B-cell count (P=0.05) were significantly higher as compared to patients who did not achieve ALC recovery by day +15. Of note, within the T-cell compartment, CD4+ central memory (P=0.02, see Figure), CD8+ central memory (P=0.05), T-regulatory (T reg) (P=0.02, CD4+25+) populations were significantly lower. The distribution of different immune cell subsets pre-ASCT was not associated with development of clinically significant infections before day +100 post-AHCT; however, the proportion of memory B cells measured at day+100 was significantly higher (p=0.02) in patients who had experienced an infection before day +100. In the present study we show that the distribution of different immune cell populations correlates with ALC recovery in MM. Moreover, we also confirmed the association of ALC recovery with prolonged PFS and OS. The increase in the relative proportions of CD4+ and CD8+ effector cell subpopulations and the decrease in CD4+ and CD8+ central memory and T-reg populations demonstrate that the pre-ASCT effector cell phenotype contributes to faster ALC recovery, and improved PFS/OS. Therapeutic strategies to alter the pre-ASCT and/or day + 100 immune phenotype may improve outcomes after ASCT. [Display omitted] [Display omitted] Disclosures:McCarthy:Janssen: Honoraria; Celgene: Consultancy.

An immunophenotypic and molecular diagnosis of composite hairy cell leukaemia and chronic lymphocytic leukaemia

Hairy cell leukaemia (HCL) and chronic lymphocytic leukaemia (CLL) are distinct clinicopathological B cell chronic lymphoproliferative disorders (B-CLPD). Both diseases have characteristic immunophenotypic and molecular features. The co-existence of two B-CLPD is perhaps more common than previously thought but a composite HCL and CLL has been rarely documented. A case is reported in which the morphology, integrated with an extensive immunophenotyping panel, and incorporation of the recently described HCL-associated BRAF V600E mutation, enabled the prompt diagnosis of composite HCL and CLL thus allowing appropriate treatment selection. This case serves to highlight the benefit of a multidisciplinary approach to the diagnosis of bi-clonal B-CLPD.

48: Immunophenotyping innate lymphoid and T helper cell populations in human asthma

Asthma is a heterogeneous disease driven by T-helper type 2 (Th2) cell-driven airway inflammation, due to the production of the pro-inflammatory cytokines IL-13, IL-5 and IL-4. Many cell types in the allergic lung produce IL-13, including T cells and type 2 innate lymphoid cells (ILC2). To identify the T helper cell subsets that contribute to disease, we immunophenotyped blood, bronchoalveolar lavage fluid (BAL) and induced sputum cells from asthmatic patients. Using 10–12 color flow cytometry, we characterized innate cells and T helper cell populations in two separate immunophenotyping panels, and correlated cell surface markers with intracellular cytokine production. To incorporate a larger number of parameters into a single immunophenotyping panel, we have started to use the cytometry by time-of-flight mass spectrometry (CyTOF) platform as an alternative for airway biospecimens. CyTOF allows us to look at upwards of 30–40 markers on every cell in each sample, including both cell surface and intracellular markers, and will help us determine the interplay among the different cell types that contribute to allergic airway disease.

Diagnostic Challenges of CD4+/CD56+/CD123+ hematological neoplasms

In the January/February 2013 edition, Maio et al describe a patient with CD4/CD56/CD123 ascribed as blastic plasmacytoid dendritic cell neoplasia (BDCN). However, since CD4/CD56/CD123 neoplasms are highly heterogeneous, the precise diagnosis requires an extensive immunophenotypic panel. 3 Although highly suggestive, the cytochemical positivity for CD4, CD56 together with CD123 in the absence of myeloperoxidase, CD3, CD2, CD5, and CD7, is not sufficient to determine the BDCN malignant nature. Despite the expression of CD123, the aforementioned phenotype could also correspond to acute myeloid dendritic cell leukemia or acute myeloid leukemia (myeloid leukemia cutis), especially with monocytic differentiation. The diagnostic work-up of these entities relies on a comprehensive antibody panel that should also include CD13, CD33, CD15, CD14, CD64, CD16, CD34, CD117, BDCA-2 (CD303), BDCA-4 (CD304), BDCA-3 (CD141) and TCL1. BDCN are phenotypically recognized by expression of specific plasmacytoid dendritic cell proteins (CD303 and CD304) in the absence or dim expression of myeloid markers. Conversely, acute myeloid dendritic cell leukemias specifically express CD141 along with some myeloid markers. By exclusion, the absence of CD303, CD304 and CD141, along with the presence of myeloid and monocytic markers, supports the diagnosis of myelomonocytic leukemia. Additional molecular tests targeting well-characterized abnormalities could serve as ancillary diagnostic tools. Although the panel herein proposed could not be entirely performed on skin biopsies, it could be easily applied by flow cytometry on circulating cells during the disseminated phase. It is clear that strong collaborative efforts are required to improve diagnosis and management of these rare diseases.

Oral tungstate (Na2WO4) exposure reduces adaptive immune responses in mice after challenge

Tungstate has been identified as a ground water contaminant at military firing ranges and can be absorbed by ingestion. In this study, C57BL6 mice were exposed to sodium tungstate (Na2WO4·2H2O) (0, 2, 62.5, 125, and 200 mg/kg/day) in their drinking water for an initial 28-day screen and in a one-generation (one-gen) model. Twenty-four hours prior to euthanasia, mice were intraperitoneally injected with Staphylococcal enterotoxin B (SEB) (20 μg/mouse) or saline as controls. After euthanasia, splenocytes and blood were collected and stained with lymphocyte and/or myeloid immunophenotyping panels and analyzed by flow cytometry. In the 28-day and one-gen exposure, statistically significant reductions were observed in the quantities of activated cytotoxic T-cells (TCTL; CD3+CD8+CD71+) and helper T-cells (TH; CD3+CD4+CD71+) from spleens of SEB-treated mice. In the 28-day exposures, CD71+ TCTL cells were 12.87 ± 2.05% (SE) in the 0 tungstate (control) group compared to 4.44 ± 1.42% in the 200 mg/kg/day (p < 0.001) group. TH cells were 4.85 ± 1.23% in controls and 2.76 ± 0.51% in the 200 mg/kg/day (p < 0.003) group. In the one-gen exposures, TCTL cells were 7.98 ± 0.49% and 6.33 ± 0.49% for P and F1 mice after 0 mg/kg/day tungstate vs 1.58 ± 0.23% and 2.52 ± 0.25% after 200 mg/kg/day of tungstate (p < 0.001). Similarly, TH cells were reduced to 6.21 ± 0.39% and 7.20 ± 0.76%, respectively, for the 0 mg/kg/day P and F1 mice, and 2.28 ± 0.41% and 2.85 ± 0.53%, respectively, for the 200 mg/kg/day tungstate P and F1 groups (p < 0.001). In delayed-type hypersensitivity Type IV experiments, tungstate exposure prior to primary and secondary antigen challenge significantly reduced footpad swelling at 20 and 200 mg/kg/day. These data indicate that exposure to tungstate can result in immune suppression that may, in turn, reduce host defense against pathogens.

Improved click chemistry demonstrating EdU cell proliferation with GFP expressing cells and R-PE based immunophenotyping. (P3299)

Abstract The current Click-iT® EdU kit, for measuring cell proliferation, cannot be use for the simultaneous detection of EdU, GFP and R-PE fluorescence due to the presence of copper and reactive oxygen species mediated damage to fluorescent proteins. We present modifications to the click reaction resulting in“copper safe” catalysis of the click reaction. Flow cytometry measurements of cell proliferation using Click-iT® EdU are demonstrated to be compatible with immunophenotyping panels using R-PE conjugates and GFP expressing proteins. The basis of the click chemistry improvement is the use of a copper (I) ligand combined with a modified dye azide detection reagent. We optimized components in the improved click reaction which best preserved R-PE fluorescence while obtaining a bright EdU signal. Jurkat cells were pulsed with EdU, labelled with PE conjugated antibodies and then fixed and permeabilized. EdU incorporation was then detected using click chemistry, demonstrating improved detection while maintaining PE signal. Additionally, BacMam transduced cells expressing GFP were treated with cell cycle inhibitors then pulsed with EdU and used to demonstrate GFP and click chemistry compatibility. The modified click reaction is an improvement over the original copper based click reactions and will further enhance the utility of EdU based cell proliferation applications in flow cytometry.

Validation of an eight parameter immunophenotyping panel in adult canines for assessment of immunotoxicity

Analysis of peripheral blood leukocyte populations by flow cytometry in adult beagles is a critical component of immunotoxicity assessment in regulated pre-clinical toxicology studies. In this study, data is presented utilizing a single panel, six-color method to simultaneously enumerate absolute cell counts and determine the relative percentage of leukocytes. A GLP validation was performed to determine intra- and inter-assay variance, inter-instrument variance, and pre- and post-fixation stability for the target populations. The results demonstrated all samples met acceptance criteria, CV values less than 25%, for all precision and stability intervals assessed. The intra and inter-assay data demonstrated the single panel method generated acceptable precision. Furthermore, stability results indicated whole blood samples and processed samples may be stored without a statistically significant difference in the data compared to samples immediately processed and analyzed after blood collection. This assay will provide researchers a more precise and efficient tool to evaluate the immunotoxic effects of a test article on canine peripheral blood leukocytes during pre-clinical drug testing.

The extended leukocyte differential count using the Cytodiff flow cytometric system reveals that higher CD16+ cytotoxic NK+T lymphocyte levels predict superior survival outcomes in patients with metastatic carcinoma

The recently developed Cytodiff flow cytometric system (Beckman Coulter, Miami, FL) enables leukocyte analysis using a single immunophenotyping panel tube composed of six markers and five colors and that can detect 16 leukocyte subpopulations. We performed a preliminary investigation of whether changes in any of 16 leukocyte differentials were associated with survival and treatment outcomes in patients with metastatic carcinoma or not. We measured 16 leukocyte differential counts using the Cytodiff flow cytometric system in peripheral blood samples from 40 patients with metastatic malignancy (27 stomach cancer and 13 lung cancer) before chemotherapy and at 15 day intervals after chemotherapy for 2 months. A higher percentage of CD16+ cytotoxic NK+T lymphocytes was found to be the only significant prognostic factor among by Cox regression analysis and a higher percentage of CD16+ cytotoxic NK+T lymphocytes (>5.0%) showed significantly longer survival outcomes by Kaplan-Meier analysis (P = 0.003). The Cytodiff system enables 16 leukocyte subpopulations in a one tube assay and also can operate with only small amounts of sample, although it cannot differentiate NK cells from T lymphocytes. Hence, the monitoring of all leukocyte subpopulations using Cytodiff flow cytometry may be a helpful prognostic tool for patients with metastatic carcinoma.

Reporte del Primer Consenso Colombiano de Citometría de Flujo para el estudio de trastornos hematológicos

th and 8 th of 2008, the First Colombian Consensus Conference for the Immunophenotyping of Hematological Malignancies was carried out in Cali, Colombia, with representatives from Colombia, Argentina, Chile, Peru and Mexico. The consensus included professionals from the laboratory field and clinicians from different institutions that routinely operate clinical flow cytometry with an emphasis on lymphoma and leukemia immunophenotyping. In this consensus medical indications for flow cytometry use in the detection of hematological malignancies, technical considerations for staining and samples preparation protocols, evaluation of diagnostic immunophenotyping panels, data acquisition and interpretation strategies were analyzed, and, finally, how to report the results.

BCR-ABL1+T-Lymphoblastic Leukemia/Lymphoma Is a Rare Type of Chronic Myelogenous Leukemia Blast Crisis and Has a Poor Prognosis

BCR-ABL1 + - lymphoblastic leukemia/lymphoma (T-LBLL) is rare and may be difficult to distinguish from the also very rare cases of T-lineage blast crisis of chronic myelogenous leukemia (CML). We report 3 cases of BCR-ABL1 + T-LBLL. In all 3 cases, T lineage was confirmed with an extensive immunophenotyping panel. The first patient was a 64-year-old woman with …

OMIP‐010: A new 10‐color monoclonal antibody panel for polychromatic immunophenotyping of small hematopoietic cell samples

Preijers, Frank W M B Huys, Erik Moshaver, Bijan United States Cytometry A. 2012 Jun;81(6):453-5. doi: 10.1002/cyto.a.22056. Epub 2012 Apr 24.

Exploring complexity

Exploring complexity

Simplified flow cytometric immunophenotyping panel for multiple myeloma, CD56/CD19/CD138(CD38)/CD45, to differentiate neoplastic myeloma cells from reactive plasma cells

BackgroundFlow cytometric immunophenotyping has been used to identify neoplastic plasma cell populations in patients with multiple myeloma (MM). Previous reports have described the use of several antigens, including CD38, CD138, CD56, CD117, CD52, CD19 and CD45, to distinguish distinct populations of plasma cells. The aim of this study was to evaluate a simplified immunophenotyping panel for MM analysis.MethodsA total of 70 patients were enrolled in the study, 62 of which were newly diagnosed with MM (untreated), whereas the remaining 8 were undergoing bone marrow assessment as part of follow-up after treatment (treated). Treated cases included 3 patients with relapse and 5 patients with persistence of MM. Multiparametric flow cytometric immunophenotyping was performed using monoclonal antibodies against CD56, CD19, CD138 (CD38), and CD45.ResultsIn differential counts, plasma cells in bone marrow (BM) accounted for 3.6-93.2% of the total nucleated cell count. The positive expression rates of CD56, CD19, CD138, and CD45 in neoplastic myeloma cells were 83.9%, 0%, 98.4%, and 37.1%, respectively, among the 62 untreated cases, and 75.0%, 0%, 87.5%, and 37.5%, respectively, among the 8 treated cases. CD19 expression of neoplastic plasma cells was negative in both untreated and treated cases.ConclusionThe simplified immunophenotyping panel, CD56/CD19/CD138(CD38)/CD45, is useful for distinguishing neoplastic myeloma cells from reactive plasma cells in clinical practice. In addition, CD19 represents the most valuable antigen for identifying neoplastic myeloma cells in patients with MM.