Multiparameter Flow Cytometric Assay Research Articles

Progressive Loss of Donor-Reactive CD4+ Effector Memory T Cells due to Apoptosis Underlies Donor-Specific Hyporesponsiveness in Stable Renal Transplant Recipients.

Following kidney transplantation, donor-specific hyporesponsiveness (DSH) may develop, defined as a lowered response of alloreactive T cells, specifically directed to donor Ag. This study aimed to characterize the nature of DSH through multiparameter flow cytometric assays measuring changes in phenotype and function of donor-reactive T cells after transplantation. This study characterized donor-reactive T cells, identified by CD137 expression, from the peripheral blood of stable human kidney transplant recipients (n = 47) before, at 3-5 y after, and >5 y after transplantation. The phenotype (T cell subset, differentiation status, and transcription factor expression) and function (proinflammatory cytokine production) of CD4+ and CD8+ donor-reactive CD137+ T cells was evaluated by both supervised and unsupervised analyses. Results demonstrated a decline in CD4+ donor-reactive T cells within the first 3-5 y after transplantation. Predominantly, the population of effector memory T cells capable of producing two or more proinflammatory cytokines was affected. This decline was strongly correlated with reduced proliferation of CD4+ T cells to donor Ag. The donor-reactive CD8+ T cells declined substantially only after >10 y. The frequency of T cells reactive to unrelated alloantigens did not alter significantly after transplantation, excluding an aspecific effect of immunosuppressive medication. After transplantation, an increase in donor Ag-induced apoptosis was found, specifically within the donor-reactive CD4+ memory T cell subsets. In conclusion, a significant decrease in donor-reactive polyfunctional effector memory CD4+ T cells underlies the development of DSH in kidney transplant recipients, which is likely mediated by specific activation-induced cell death.

Rac1/pSTAT3 expression: A pharmacodynamic marker panel as a first step toward optimization of thiopurine therapy in inflammatory bowel disease patients.

Thiopurine derivatives, such as azathioprine and mercaptopurine, are standard conventional treatment options in inflammatory bowel disease (IBD). Unfortunately, approximately half of patients discontinue thiopurine therapy within 2 years. To improve the prediction of clinical effectiveness, thiopurine therapy is currently optimized using therapeutic drug monitoring. Ras-related C3 botulinum toxin substrate 1 (Rac1) has been suggested as a potential pharmacodynamic marker of the thiopurine effect in lymphocytes. The active thiopurine metabolite 6-thioguanine triphosphate (6-Thio-GTP) causes T cell apoptosis via Rac1 and the downstream transcription factor signal transducer and activator of transcription 3 (STAT3). The aim of this study was to develop and validate a functional pharmacodynamic multiparameter flow cytometric assay to determine Rac1/pSTAT3 expression in the various leukocyte subpopulations in peripheral blood in order to predict therapeutic response in IBD patients in the future. Peripheral blood samples of healthy subjects (no fever or clinical complaints of active disease, C-reactive protein < 10mg/L) were used for immunocytochemical labeling, applying an optimized fixation and permeabilization strategy. A gating procedure was performed to separate all leukocyte subpopulations. Quantitative data were obtained by measuring presence and median fluorescent intensity. In vitro, Rac1 presence and expression were detectable in all leukocyte subpopulations. After IL-6 stimulation, used as proxy for inflammation, a distinct pSTAT3 signal could be detected in T lymphocytes of healthy subjects. In vivo, an upregulated pSTAT3 signal was detected in nearly all IBD patients with active disease and differed substantially from the signal found in IBD patients in remission on thiopurines and healthy subjects. We developed and validated a functional flow cytometric assay to assess Rac1 and pSTAT3 presence and expression. This opens a venue for a pharmacodynamic assay to predict thiopurine effectiveness in IBD patients.

Frequency and Clinical Significance of the Expression of the Multidrug Resistance Proteins MDR1/P-Glycoprotein, MRP1, and LRP in Acute Myeloid Leukemia. A Southwest Oncology Group Study

Therapeutic resistance is a major obstacle in the treatment of acute myeloid leukemia (AML). Such resistance has been associated with rapid drug efflux mediated by the multidrug resistance gene 1 (MDR1; encoding P-glycoprotein) and more recently with expression of other novel proteins conferring multidrug resistance such as MRP1 (multidrug resistance-associated protein 1) and LRP (lung resistance protein). To determine the frequency and clinical significance of MDR1, MRP1, and LRP in younger AML patients, we developed multiparameter flow cytometric assays to quantify expression of these proteins in pretreatment leukemic blasts from 352 newly diagnosed AML patients (median age, 44 years) registered to a single clinical trial (SWOG 8600). Protein expression was further correlated with functional efflux by leukemic blasts [assessed using two substrates: Di(OC)(2) and Rhodamine 123] and with the ability of MDR-reversing agents to inhibit efflux in vitro. MDR1/P-glycoprotein expression, which was highly correlated with cyclosporine-inhibited efflux, was noted in only 35% of these younger AML patients, distinctly lower than the frequency of 71% we previously reported in AML in the elderly (Blood 89:3323, 1997). Interestingly, MDR1 expression and functional drug efflux increased with patient age, from a frequency of only 17% in patients less than 35 years old to 39% in patients aged 50 years (P =.010). In contrast, MRP1 was expressed in only 10% of cases and decreased with patient age (P =. 024). LRP was detected in 43% of cases and increased significantly with increasing white blood cell counts (P =.0015). LRP was also marginally associated with favorable cytogenetics (P =.012) and French-American-British (FAB) AML FAB subtypes (P =.013), being particularly frequent in M4/M5 cases. Only MDR1/P-glycoprotein expression and cyclosporine-inhibited efflux were significantly associated with complete remission (CR) rate (P(MDR1) =.012; P(efflux) =.039) and resistant disease (RD; P(MDR1) =.0007; P(efflux) =.0092). No such correlations were observed for MRP1 (P(CR) =.93; P(RD) =.55) or LRP (P(CR) =.50; P(RD) =.53). None of these parameters were associated with overall or relapse-free survival. Unexpectedly, a distinct and nonoverlapping phenotype was detected in 18% of these cases: cyclosporine-resistant efflux not associated with MDR1, MRP1, or LRP expression, implying the existence of other as yet undefined efflux mechanisms in AML. In summary, MDR1 is less frequent in younger AML patients, which may in part explain their better response to therapy. Neither MRP1 nor LRP are significant predictors of outcome in this patient group. Thus, inclusion of MDR1-modulators alone may benefit younger AML patients with MDR1(+) disease.

Determination of the proliferative fractions in differentiating hematopoietic cell lineages of normal bone marrow.

Because of the proven prognostic value of Ki-67 as a proliferation marker in several types of solid cancers, our goal is to develop and validate a multiparameter flow cytometric assay for the determination of the Ki-67 expression in hemato-oncological diseases. The aim of the present study was to establish the reference values for the fraction of Ki-67 positive cells in and during maturation of individual hematopoietic cell lineages present in normal bone marrow. Aspirates derived from femoral heads of 50 patients undergoing a hip replacement were used for the flow cytometric quantification of Ki-67 expression in the different hematopoietic cell populations of healthy bone marrow. Furthermore, the proliferative index was investigated in detail for the maturation steps during erythro-, myelo-, and monopoiesis using recently described immunophenotypic profiles in combination with a software-based maturation tool. Reference values for the proliferative index were established for different relevant hematopoietic cell populations in healthy bone marrow. During maturation, the size of the Ki-67 positive fraction was the highest in the most immature compartment of the myeloid, monocytic, and erythroid cell lineages, followed by a steady decline upon cell maturation. While proerythroblasts showed a proliferative activity of almost 100%, the myelo- and monoblast showed a lower proliferative index of on average of 50%, indicating that a relatively large proportion of these cells exist in a quiescent state. In conclusion, we can state that when using a novel combination of immunophenotypic markers, the proliferation marker (Ki-67) and a software-based maturation tool, it was possible to determine the proliferative fractions in the diverse hematopoietic cell lineages in bone marrow, in particular during maturation. Using this approach, the proliferative indices for the normal myelo-, mono-, and erythropoiesis were determined, which can be used as a reference in future studies of hematologic malignancies originating from bone marrow. © 2018 International Society for Advancement of Cytometry.

Development and validation of a rapid, aldehyde dehydrogenase bright–based cord blood potency assay

AbstractBanked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDHbr]), along with viable CD45+ or CD34+ cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDHbr, CD34+, and CFU content of 3908 segments over a 5-year period. ALDHbr (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34+ (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDHbr content of the CBU. These results suggest that the ALDHbr segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation.

Lovastatin Decreases the Expression of CD133 and Influences the Differentiation Potential of Human Embryonic Stem Cells.

The lipophilic statin lovastatin decreases cholesterol synthesis and is a safe and effective treatment for the prevention of cardiovascular diseases. Growing evidence points at antitumor potential of lovastatin. Therefore, understanding the molecular mechanism of lovastatin function in different cell types is critical to effective therapy design. In this study, we investigated the effects of lovastatin on the differentiation potential of human embryonic stem (hES) cells (H9 cell line). Multiparameter flow cytometric assay was used to detect changes in the expression of transcription factors characteristic of hES cells. We found that lovastatin treatment delayed NANOG downregulation during ectodermal and endodermal differentiation. Likewise, expression of ectodermal (SOX1 and OTX2) and endodermal (GATA4 and FOXA2) markers was higher in treated cells. Exposure of hES cells to lovastatin led to a minor decrease in the expression of SSEA-3 and a significant reduction in CD133 expression. Treated cells also formed fewer embryoid bodies than control cells. By analyzing hES with and without CD133, we discovered that CD133 expression is required for proper formation of embryoid bodies. In conclusion, lovastatin reduced the heterogeneity of hES cells and impaired their differentiation potential.

What Is the Frequency of Transplant-Eligible Multiple Myeloma Patients Being Cured? the Impact of an MGUS-like Signature at Diagnosis and MRD-Negativity

Introduction: Although multiple myeloma (MM) is typically described as an incurable disease, it has been shown in recent years that a small fraction of patients may reach more than 10-years progression-free survival (PFS), which is considered as the minimum threshold to identify patients in "operational cure". However, because of the scarcity of available data there is significant lack of knowledge in MM regarding the frequency of cases attaining operational cure, nor the existence of biomarkers that could prospectively predict such curability.Methods: Herein, we sought to define the frequency as well as the biomarkers predictive of operational cure in a large series of uniformly-treated transplant-eligible patients enrolled in the PETHEMA/GEM2000 protocol (VBMCP/VBAD followed by HDT/ASCT and 2 years of maintenance with interferon and prednisone). Patients' follow-up was updated at the time of abstract submission, and the median follow-up of the series is now of 12-years. We used an automated multiparameter flow cytometric (MFC) classification model focused on the analysis of the bone marrow plasma-cell compartment to identify among newly diagnosed symptomatic MM those with MGUS-like vs. MM-like phenotypic signatures. Minimal residual disease (MRD) was monitored using a first-generation 4-color MFC assay (CD38-FITC / CD56-PE / CD19-PerCPCy5.5 / CD45-APC) with a limit of detection of 10-4. PFS and overall survival (OS) were measured from the time of diagnosis.Results: From a total of 1075 patients enrolled in the GEM2000 protocol, 763 were eligible for this analysis because they either relapsed or died during the first 10-years from diagnosis (n=666; 87%), or remained progression-free and alive for more than 10-yers (n=97; 13%); accordingly, all patients remaining progression-free and alive but for which the follow-up was inferior to 10-years were excluded from the analysis. We then investigated the biomarkers that could help to identify patients reaching operational cure after HDT/ASCT. As compared to the vast majority of cases, patients reaching >10-years PFS (13%) had significantly less frequent anemia (76% vs. 60%, respectively; P=.002), as well as more frequent Durie-Salmon stage IA (14% vs. 6%; P=.004), MGUS-like signature as determined by the automated MFC algorithm (28% vs. 6%; P<.001), complete response (CR) after HDT/ASCT (51% vs. 35%; P=.003), and MRD-negativity by MFC (72% vs. 31%; P<.001). Other biomarkers such as ISS, LDH, ploidy and proliferation were not significantly different among patients reaching >10-years PFS vs. those who relapsed earlier. On multivariate analysis, only the presence of an MGUS-like signature at baseline (P=.04; HR: 3.9) and MRD-negativity at day+100 after HDT/ASCT (P=.006; HR: 6.3) emerged as independent predictive markers for >10-years PFS; anemia, Durie-Salmon and CR status were not retained in the logistic regression model. Accordingly, patients with an MFC-defined baseline MGUS-like signature reaching MRD-negativity after HDT/ASCT (n=14) had a median PFS of 10-years and a 10-year OS rate of 79%, which were significantly superior to those observed among cases with MM-like signatures being MRD-negative (n=54) or positive (n=99) after therapy [median PFS of 6 and 3 years (P<.001); 10-year overall survival rates of 55% and 19% (P<.001)].Conclusions: We demonstrated that operational cure (i.e.: >10-years PFS) was possible for 13% of transplant-eligible MM patients before the era of novel agents. Curability rates were particularly frequent among patients with a benign phenotypic signature at diagnosis and MRD negativity after HDT/ASCT, suggesting a remarkable clinical benefit of attaining deep remissions after intensive treatment for patients with early MM. DisclosuresPaiva:Sanofi: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Onyx: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy; Celgene: Consultancy. Puig:The Binding Site: Consultancy; Janssen: Consultancy. Mateos:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria; Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: Honoraria.

Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer.

ObjectivesPARP inhibitors (PARPi) are a novel class of drugs with activity in patients with acquired or germline homologous recombination (HR) deficiency-associated high-grade serous ovarian cancer (HGSOC). We hypothesized that measuring γH2AX as an indicator of DNA double-strand breaks (DSB), and MRE11 or RAD51 as an indicator of DSB repair, would reflect HR status and predict response to PARPi-based therapy. Our aim was to develop and use high-throughput multiparametric flow cytometry to quantify γH2AX with MRE11 or RAD51 in PBMCs as a readily available surrogate.MethodsHealthy donor PBMCs were used for assay development and optimization. We validated induction of γH2AX, MRE11 and RAD51 by staining with fluorophore-conjugated antibodies. The multiparameter flow cytometric method was applied to PBMC samples from recurrent HGSOC patients who were treated with PARPi, olaparib and carboplatin.ResultsStimulation was necessary for quantification of a DNA damage response to olaparib/carboplatin in healthy donor PBMCs. The flow cytometric protocol could not distinguish between cytoplasmic and nuclear RAD51, erroneously indicating activation in response to injury. Thus, MRE11 was selected as the marker of DSB repair. PBMCs from 15 recurrent HGSOC patients were then examined. Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5–13.2] v. 3.3 [2.8–9.9], p < 0.03) compared with responders.ConclusionsWe successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs. Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0604-z) contains supplementary material, which is available to authorized users.

SOX2 Is Regulated Differently from NANOG and OCT4 in Human Embryonic Stem Cells during Early Differentiation Initiated with Sodium Butyrate

Transcription factors NANOG, OCT4, and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however, their expression profiles during early differentiation of hES cells are unclear. In this study, we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG, OCT4, and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells, characterized by specific expression patterns of NANOG, OCT4, and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time, indicating a gradual mode of developmental transition in these cells. Notably, distinct regulation of SOX2 during early differentiation events was detected, highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation.

Addition of interferon‐α to the p53‐SLP® vaccine results in increased production of interferon‐γ in vaccinated colorectal cancer patients: A phase I/II clinical trial

We previously established safety and immunogenicity of a p53 synthetic long peptides (p53-SLP®) vaccine. In the current trial, we investigated whether combination of interferon-alpha (IFN-α) with p53-SLP® is both safe and able to improve the induced p53-specific IFN-γ response. Eleven colorectal cancer patients successfully treated for metastatic disease were enrolled in this study. Of these, nine patients completed follow-up after two injections with p53-SLP® together with IFN-α. Safety and p53-specific immune responses were determined before and after vaccination. Furthermore, cryopreserved PBMCs were compared head-to-head to cryopreserved PBMCs obtained in our previous trial with p53-SLP® only. Toxicity of p53-SLP® vaccination in combination with IFN-α was limited to Grade 1 or 2, with predominantly small ongoing swellings at the vaccination site. All patients harbored p53-specific T cells after vaccination and most patients showed p53-specific antibodies. Compared to the previous trial, addition of IFN-α significantly improved the frequency of p53-specific T cells in IFN-γ ELISPOT. Moreover, in this trial, p53-specific T cells were detectable in blood samples of all patients in a direct ex vivo multiparameter flowcytometric assay, opposed to only 2 of 10 patients vaccinated with p53-SLP® only. Finally, patients in this trial displayed a broader p53-specific immunoglobulin-G response, indicating an overall better p53-specific T-helper response. Our study shows that p53-SLP® vaccination combined with IFN-α injection is safe and capable of inducing p53-specific immunity. When compared to a similar trial with p53-SLP® vaccination alone the combination was found to induce significantly more IFN-γ producing p53-specific T cells.

A novel multiparameter flow cytometric assay for inosine triphosphatase expression analysis in leukocytes

The aim of this study was to assess inosine triphosphate (ITPase) expression in the different leukocyte populations present in peripheral blood samples of a nonimmune compromised control group. For this purpose, a multiparameter flow cytometric assay was developed and performed to study ITPase expression in peripheral leukocyte subpopulations of healthy volunteers (n = 20). Qualitative ITPase expression was assessed by determining the percentage of ITPase-positive cells. Quantitative data were obtained by measuring the median fluorescent intensity (MFI). Subcellular localization of ITPase was analyzed using immunocytochemistry. Immunocytochemistry showed that ITPase is present in all leukocytes and localized intracellular. Based on this finding, a multiparameter flow cytometric assay was developed using a Fix & Perm strategy. Qualitative and quantitative ITPase expression remained stable (variation, <10%) for at least 48 h after blood sampling. MFI values showed that activated monocytes contained significantly more ITPase when compared to the total monocyte fraction (P < 0.0001), which subsequently had a higher amount of expression than granulocytes (P < 0.0001). In addition, the phagocyte subpopulations ([activated] monocytes and granulocytes) contained significantly higher levels of ITPase when compared to lymphocytes (P < 0.0001). Within the lymphocyte fraction, it appeared that T-helper cells contained significantly higher ITPase levels when compared to cytotoxic T cells, B lymphocytes, and natural killer cells (P < 0.0001). Our study is the first which describes a flow cytometry assay to analyze ITPase expression in leukocytes qualitatively as well as quantitatively and visualizes the intracellular localization of ITPase in leukocytes. © 2012 International Society for Advancement of Cytometry.

A recombinant adenovirus expressing immunodominant TB antigens can significantly enhance BCG-induced human immunity

Despite the availability of Bacille Calmette Guérin (BCG) vaccines, Mycobacterium tuberculosis currently infects billions of people and millions die annually from tuberculosis (TB) disease. New TB vaccines are urgently needed. We studied the ability of AERAS-402, a recombinant, replication-deficient adenovirus type 35 expressing the protective M. tuberculosis antigens Ag85A, Ag85B, and TB10.4, to boost BCG immunity in an area of low TB endemicity. In volunteers primed with BCG 3 or 6 months prior to AERAS-402 boosting, significant CD4(+) and CD8(+) T cell responses were induced. Ag85-specific responses were more strongly boosted than TB10.4-specific responses. Frequencies of TB-specific CD8(+) T cells reached>50 fold higher than pre-AERAS boosting levels, remarkably higher than reported in any previous human TB vaccine trial. Multiparameter flow cytometric assays demonstrated that AERAS-402-boosted CD4(+) and CD8(+) T cells were multifunctional, producing multiple cytokines and other immune effector molecules. Furthermore, boosted T cells displayed lymphoproliferative capacity, and tetramer analyses confirmed that antigen-specific CD8(+) T cells were induced. BCG and AERAS-402 vaccinations given 3 and 6 months apart appeared equivalent. Our results indicate that AERAS-402 is a promising TB vaccine candidate that can significantly enhance both CD4(+) and CD8(+) TB-specific T cell responses after BCG priming. ClinicalTrials.gov Identifier: NCT01378312.

Flow cytometric chemosensitivity assay as a predictive tool of early clinical response in acute lymphoblastic leukemia

Residual disease or rapidity of response to induction therapy is among the most powerful predictors of outcome in pediatric acute lymphoblastic leukemia (ALL). Utilizing a multiparameter flow cytometric chemosensitivity assay (FCCA), we studied the relationship between in vitro drug sensitivity of diagnostic leukemic blasts from 30 children with ALL and rapidity of response to induction therapy. We also analyzed the in vitro drug sensitivity of de novo leukemic blasts among various clinical subsets. Compared to rapid early responders (RERs), slow early responders (SERs) had a significantly greater in vitro drug resistance to dexamethasone (DEX; P = 0.04) and prednisone (P = 0.05). The studies with all other drugs showed a non-significant trend with the SER having a higher in vitro drug resistance compared to the RER. Risk group stratified analyses indicated that in vitro resistance to asparaginase (ASP), DEX, and vincristine (VCR) were each significantly related to having very high risk ALL. Additionally, a significantly higher in vitro drug resistance to ASP and VCR was associated with unfavorable lymphoblast genetics and ultimate relapse. Our data indicate that this FCCA is a potentially simple and rapid method to detect inherent resistance to initial ALL therapy very early in induction, thus allowing for treatment modification shortly thereafter.

Measuring Angiogenic Cytokines, Circulating Endothelial Cells, and Endothelial Progenitor Cells in Peripheral Blood and Cord Blood: VEGF and CXCL12 Correlate with the Number of Circulating Endothelial Progenitor Cells in Peripheral Blood

Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) are thought to play an important role in the vascularization of damaged tissues and cancers. These cells are also required for tissue-engineered blood vessels and to help skin substitutes revascularize more efficiently. A standard approach to the phenotyping and enumeration of CEC and EPC is key to the development of new therapies, and the identification of biomarkers within the blood that regulate their levels may be important for the treatment of cancer. We have devised an improved multiparameter flow cytometric assay for CEC and circulating EPC enumeration. This assay uses antibodies recognizing CD133 and CD34 to identify EPC and CEC, respectively, and incorporates specific markers CD144 and vascular endothelial growth factor receptor 2 (VEGFR-2) for both CEC and EPC cells. In peripheral blood (PB), mean CEC numbers were 55 +/- 95 mL(-1) and mean EPC numbers were 44 +/- 58 mL(-1) (n = 60). We also found a significant correlation of both plasma VEGF (r = 0.90, p < 0.001) and CXCL12 (r = 0.84, p < 0.001) with EPCs, but not CECs. The cytokines also correlated with each other (r = 0.85, p < 0.001). In umbilical cord blood (UCB) we found on average 13 times more CEC (719 +/- 338 mL(-1)) and 7 times more EPC (299 +/- 245 mL(-1)) than in PB. However, serum VEGF and CXCL12 levels in UCB did not correlate with either EPC or CEC numbers. These results suggest a major role for VEGF and CXCL12 in the control of marrow-derived EPCs in adult PB and provide normal data for comparison with patient populations.

An analytical workflow for investigating cytokine profiles

Understanding cytokine profiles of disease states has provided researchers with great insight into immunologic signaling associated with disease onset and progression, affording opportunities for advancement in diagnostics and therapeutic intervention. Multiparameter flow cytometric assays support identification of specific cytokine secreting subpopulations. Bead-based assays provide simultaneous measurement for the production of ever-growing numbers of cytokines. These technologies demand appropriate analytical techniques to extract relevant information efficiently. We illustrate the power of an analytical workflow to reveal significant alterations in T-cell cytokine expression patterns in type 1 diabetes (T1D) and breast cancer. This workflow consists of population-level analysis, followed by donor-level analysis, data transformation such as stratification or normalization, and a return to population-level analysis. In the T1D study, T-cell cytokine production was measured with a cytokine bead array. In the breast cancer study, intracellular cytokine staining measured T cell responses to stimulation with a variety of antigens. Summary statistics from each study were loaded into a relational database, together with associated experimental metadata and clinical parameters. Visual and statistical results were generated with custom Java software. In the T1D study, donor-level analysis led to the stratification of donors based on unstimulated cytokine expression. The resulting cohorts showed statistically significant differences in poststimulation production of IL-10, IL-1 beta, IL-8, and TNF beta. In the breast cancer study, the differing magnitude of cytokine responses required data normalization to support statistical comparisons. Once normalized, data showed a statistically significant decrease in the expression of IFN gamma on CD4+ and CD8+ T cells when stimulated with tumor-associated antigens (TAAs) when compared with an infectious disease antigen stimulus, and a statistically significant increase in expression of IL-2 on CD8+ T cells. In conclusion, the analytical workflow described herein yielded statistically supported and biologically relevant findings that were otherwise unapparent.

In Vitro Drug Sensitivity as a Predicitive Tool of Early Clinical Response in Childhood Acute Lymphoblastic Leukemia.

Background: Residual disease or rapidity of response to induction therapy is among the most powerful predictors of outcome in pediatric Acute Lymphoblastic Leukemia (ALL). Various methods to determine response during induction have been in use in clinical investigation. We hypothesize that drug sensitivity at the cellular level predicts rapidity of response to induction therapy in ALL. We recently developed a high resolution flow cytometry based cytotoxicity assay for in vitro cellular drug response profiling for pediatric ALL. This method has a turnaround time of 48 hours and the ability to measure drug effect specific to leukemic cells regardless of number of admixed normal cells. We report preliminary data that correlate results of this drug sensitivity assay with rapidity of response to induction therapy among patients with ALL.Methods: We performed in vitro tests, applying a multiparameter flow cytometric drug cytotoxicity assay, on bone marrow (BM) samples of 23 patients with newly diagnosed standard (n = 10), high (n = 11), and very high (n = 2) risk ALL, as defined by the Children's Oncology Group (COG) and NCI risk classification. Fourteen patients were rapid early responders (RER) and 9 were slow early responders (SER) by COG criteria at day 15 and 29. Cryopreserved cells from BM samples were thawed and determined to have adequate viability by trypan blue dye exclusion. Drugs were tested at three different concentrations, each in triplicate. Concentrations tested were based on an empirically derived cut-off concentration (EDCC) adopted from published in vitro studies, chosen to produce a large scatter of survival index values among samples. Leukemic blasts were specifically identified by surface markers, CD 45, CD 19 and CD 10 or CD 3, while cytotoxicity was measured with Annexin V based apoptosis. Leukemic cell survival index (LCSI = Average Replicate /Average Control x 100) was determined at 48 hours after in vitro exposure to individual standard induction agents for pediatric ALL: vincristine, asparaginase, dexamethasone, prednisone and daunomycin. LCSI differences between RER and SER were compared using Wilcoxon rank sum test for each drug and concentration. The mixed effects model was used to evaluate the overall difference of LCSI between RER and SER over the three concentrations (referred to as “averaged concentrations”).Results: For dexamethasone, a significantly lower LCSI was seen in the RER compared with the SER cohort at individual and averaged concentrations: RER mean LCSI = 40.2%, SER mean LCSI = 70.1% (p = 0.01, mixed effects model). A trend toward a lower mean LCSI in the RER compared with the SER group was noted for asparaginase and vincristine at individual and averaged concentrations (p < 0.1). Mean LCSI was not different between the RER and SER groups for daunomycin and prednisone at individual or averaged concentrations.Conclusions: This in vitro drug sensitivity assay provides a response profile for dexamethasone that correlates with in vivo response to induction therapy. Research is ongoing with more patient samples in order to achieve a greater statistical power to detect a smaller difference for all drugs tested. Further research will also correlate clinical response with LCSI using drug combinations in vitro. Results of these studies will determine the potential value of this assay for early risk stratification and modification of therapy in de novo or relapsed ALL.

Drug resistance in B‐cell chronic lymphocytic leukemia: Predictable by in vitro evaluation with a multiparameter flow cytometric cytotoxicity assay

Patients with B-cell chronic lymphocytic leukemia (B-CLL) often demonstrate variable responses to similar treatments. It would be highly desirable to develop a personalized therapeutic strategy for selection of appropriate drugs or regimens based on the drug sensitivity profiles of leukemic cells from individuals. We applied a multiparameter flow cytometric drug cytotoxicity assay to evaluate drug effects specifically on B-CLL cells from 43 individuals after leukemic cells were incubated in vitro with fludarabine, chlorambucil, cladribine, or prednisolone. We demonstrated that different B-CLL cell populations from 43 individuals showed a marked variability in drug sensitivity. In vitro resistance to fludarabine was greatest in B-CLL cells with deletions of p53, a cytogenetic abnormality that is almost invariably associated with a poor therapeutic response clinically. In vitro drug sensitivity profiles analyzed by a multiparameter flow cytometric cytotoxicity assay may serve as a tool to facilitate individualized selection of appropriate drugs for treatment in B-CLL. Prospective trials will be needed to validate the clinical utility of this flow cytometric cytotoxicity assay.

A Novel Multiparameter Flow Cytometric Cytotoxicity Assay Detects p53 Deletion as the Major Cause of In Vitro Resistance to Fludarabine in B-Cell Chronic Lymphocytic Leukemia.

Individual patients with B-CLL demonstrate variable responses to standard induction and salvage therapeutic regimens. It would be highly desirable to develop a predictable and reproducible laboratory diagnostic strategy that guides the selection of appropriate drugs and/or regimens based on the drug sensitivity and resistance profiles of leukemic cells for individual patients. As a first step towards this goal, a study was designed to investigate the differences of in vitro drug sensitivity profiles of leukemic cells with different cytogenetic abnormalities from CLL patients. CLL cells from 43 patients were incubated in vitro with four commonly used chemotherapeutic agents (fludarabine, chlorambucil, cladribine or prednisolone) individually or in combination. Multiparameter flow cytometry was utilized to determine the decrease in leukemic cell viability after drug exposure. Both fresh and cryopreserved samples were assessed and were found to be equivalent for assay, regardless of the percentage of B-CLL cells or the degree of spontaneous apoptosis. The highest in vitro resistance to fludarabine, was seen in all seven cases of B-CLL cells with deletions of p53, a cytogenetic abnormality associated with poor clinical outcome. Interestingly, in vitro response to chlorambucil and prednisolone was seen some CLL cases with p53 deletion and correlated with clinical response to these drugs. In CLL cases without p53 deletion, a marked variability in vitro drug sensitivity CLL cells was observed but no significant difference was detected among cases with normal cytogenetics (n=13), ATM deletion (n=4), trisomy 12 (n=3), or 13q deletion (n=7). Our findings provide direct evidence of cellular resistance to fludarabine in CLL associated with p53 deletion, confirming prior clinical observations. In vitro drug sensitivity assay may prove useful in guiding choices for therapy for CLL patients based on the drug sensitivity profile of leukemic cells in individuals.

A New Multi-Parameter Flow Cytometric Assay for Monitoring Lymphoma Growth and Spread in a Pre-Clinical Murine Model for Human Lymphoma.

Murine lymphoma models are commonly used to test the efficacy of idiotype-based vaccines for B-cell lymphoma prior to human trials. Mouse survival has been used as an indirect measure of the tumor response to the vaccine. In this study, we describe a multiparameter flow cytometric (MPFC) assay that enables direct assessment of the tumor at the inoculation site and detection of lymphoma metastases. In initial experiments, we show that 38C13 mouse lymphoma cells expressed an aberrant phenotype compared to normal splenocytes. The combination of CD45, CD19, B220 and an anti-idiotypic antibody (S1C5) along with a sequential gating technique defined the 38C13 cells with high sensitivity and specificity. The specificity of the MPFC assay for 38C13 lymphoma cells was greater than a gating strategy using light scatter and idiotype antibody binding. The MPFC assay was used to monitor 38C13 tumor kinetics in the C3H/HeN mouse and demonstrated tumor growth at the inoculation site and metastases to the lymph nodes and bone marrow. The presence of 38C13 metastases in lymphoid organs and bone marrow predicted by the MPFC assay were confirmed by histology and immunohistochemistry (IHC). The MPFC assay will enable investigators to monitor 38C13 lymphoma tumor response to individual vaccines or other lymphoma therapy prior to clinical trials. Furthermore, the MPFC concept could be readily applied to other models of human B cell lymphoma.

A new multi-parameter flow cytometric assay for monitoring lymphoma growth and spread in a pre-clinical murine model for human lymphoma.

Mouse survival is commonly used as an indirect measure of lymphoma tumor response to anti-idiotype vaccine; however, this gives no information regarding residual lymphoma cells at primary or metastatic sites. We aimed to develop a method with which to monitor lymphoma tumor kinetics in the mouse as an independent measure of vaccine efficacy. We developed a multi-parameter flow cytometric (MPFC) assay for 38C13 mouse lymphoma cells using sequential gating to detect aberrant antigen expression and binding by an anti-idiotype antibody (S1C5). Subsequently, we tested the utility of the MPFC assay in a 38C13 tumor modeling study in the C3H/HeN mouse. The MPFC assay was demonstrated in vitro to have both high specificity and sensitivity for 38C13 lymphoma cells. In tumor kinetic studies in the C3H/HeN mouse, as the tumor enlarged, the MPFC assay showed increasing prevalence of 38C13 cells at the inoculation site in addition to metastases to lymphoid organs and bone marrow. The latter findings were confirmed by both histology and immunohistochemistry. The MPFC assay is an independent parameter for monitoring 38C13 lymphoma kinetics and could be used to monitor tumor response to individual vaccines or other lymphoma therapy prior to clinical trials.