Flow Cytometric Techniques Research Articles

Use of flow cytometry in the detection of minimal residual disease in haematological malignancy

Minimal residual disease (MRD) post-treatment remains the major barrier to long-term survival in many haematological malignancies. Detection of MRD has the potential to improve long-term survival through identification of those patients who may require intensification of treatment. The ability of flow cytometric techniques in the detection of MRD has been improved through the capacity to count more cells, use of more antibodies in defining the immunophenotype of each cell and development of protocols for detection of MRD. Detection of MRD by flow cytometry in childhood B-ALL post-induction has been demonstrated to be a valuable prognostic indicator and has been used as a guide to therapy in a number of recent trials. Development of a protocol for detection of MRD in B-ALL will be discussed. Recently, consensus guidelines have been developed by the European Research Initiative on CLL. The uptake by Australian laboratories of the consensus will be discussed. Detection of MRD in acute myeloid leukaemia remains problematic in view of the complexity of the leukaemia associated pheno-types, the heterogeneity of disease and the absence of well defined protocols. The detection of MRD by flow cytometry is becoming important in the assessment of response in B-ALL and CLL, however the place detection of MRD by flow cytometry in AML remains unclear.

A look into the future: can minimal residual disease guide therapy and predict prognosis in chronic lymphocytic leukemia?

Over the past 2 decades, dramatic improvements in the efficacy of treatments for chronic lymphocytic leukemia have led to progressively higher percentages of clinical complete remissions. A molecular eradication of the leukemia has become not only a desirable, but also an achievable, end point that needs to be evaluated within clinical trials. The assessment of complete remission only at the clinical and morphological level is insufficient, at least for physically fit patients. The detection of minimal residual disease (MRD) in chronic lymphocytic leukemia has become feasible using PCR-based or flow cytometric techniques that reproducibly allow reaching the detection level of less than 1 leukemic cell per 10 000 leukocytes (10(-4)), the level currently defined as MRD(-) status. Emerging data indicate that the MRD status during and at the end of treatment is one of the most powerful predictors of progression-free and overall survival. This predictor appears to be independent of clinical response, type or line of therapy, and known biological markers. For these reasons, the time is ripe to test the use of MRD as a surrogate marker of clinical end points and as a real-time marker of efficacy and/or resistance to the administered therapies. In the near future, clinical trials will determine whether MRD assessment can be used for guiding therapy, either to improve quality of responses through consolidation or to prevent relapses through preemptive therapies based on the reappearance of MRD.

A look into the future: can minimal residual disease guide therapy and predict prognosis in chronic lymphocytic leukemia?

Abstract Over the past 2 decades, dramatic improvements in the efficacy of treatments for chronic lymphocytic leukemia have led to progressively higher percentages of clinical complete remissions. A molecular eradication of the leukemia has become not only a desirable, but also an achievable, end point that needs to be evaluated within clinical trials. The assessment of complete remission only at the clinical and morphological level is insufficient, at least for physically fit patients. The detection of minimal residual disease (MRD) in chronic lymphocytic leukemia has become feasible using PCR-based or flow cytometric techniques that reproducibly allow reaching the detection level of less than 1 leukemic cell per 10 000 leukocytes (10−4), the level currently defined as MRD− status. Emerging data indicate that the MRD status during and at the end of treatment is one of the most powerful predictors of progression-free and overall survival. This predictor appears to be independent of clinical response, type or line of therapy, and known biological markers. For these reasons, the time is ripe to test the use of MRD as a surrogate marker of clinical end points and as a real-time marker of efficacy and/or resistance to the administered therapies. In the near future, clinical trials will determine whether MRD assessment can be used for guiding therapy, either to improve quality of responses through consolidation or to prevent relapses through preemptive therapies based on the reappearance of MRD.

Erythropoietin-Induced Erythroid Precursor Pool Depletion Causes Erythropoietin Hyporesponsiveness

The purpose of this study is to demonstrate that the erythroid precursor depletion in bone marrow induced by recombinant human erythropoietin (rHuEPO) treatment may be another contributing factor to erythropoietin hyporesponsiveness. Healthy Wistar rats were given single dose (SD) or multiple doses (MD) of rHuEPO (100 IU/kg). In MD study, animals were challenged with thrice-weekly over two weeks. Blood, bone marrow and spleen (for SD only) were collected. The erythropoietic responses in bone marrow and spleen were quantified using a flow cytometric immunophenotyping technique. A mathematical approach involving measuring reticulocyte age distribution was developed to evaluate the reticulocyte loss due to neocytolysis. A reduced level of erythropoietic responses below the baseline was observed for both MD and SD studies. In SD study, the reticulocyte decreased below the baseline after day 6. A depletion of the bone marrow erythroid precursor cells was observed. However, neocytolysis of reticulocyte only occurs from day 3-5 after rHuEPO injection. The findings demonstrate that EPO-induced erythroid precursor depletion in bone marrow is responsible for reduced reticulocyte response and may contribute to erythropoietin hyporesponsiveness. Therefore, this study provides further justification for reducing the doses of erythropoietin-stimulating agents in anemic patients demonstrating hyporesponsiveness.

Analysis of Early Disease Response in Childhood ALL: Can Peripheral Blood Replace Bone Marrow Analysis?.

Abstract 2523▪▪This icon denotes a clinically relevant abstractThe detection of minimal residual disease (MRD) is highly predictive of outcome in pediatric acute lymphoblastic leukemia (ALL) and has now been integrated into the majority of treatment regimens. Early studies demonstrated the strong prognostic value of post-induction MRD measurement, using both molecular and flow cytometric techniques, allowing treatment intensity to be tailored to response. More recent studies have examined the value of MRD measurement at earlier time points and using peripheral blood (PB) in place of bone marrow (BM) in an attempt to reduce invasive procedures.Within the United Kingdom (UK), previous national trials have shown that post-induction MRD assessment, using molecular techniques, is highly predictive of relapse, allowing treatment stratification based on MRD levels. We have previously demonstrated the value of flow cytometric MRD assessment at early time points in response prediction (Motwani et al, Blood 2007a), however, as yet there has been no assessment of MRD in peripheral blood at early time points using flow cytometric methods in the context of a UK national trial.To address this we assessed MRD by flow cytometry using Day 8 (D8) and/or D15 paired PB and BM samples taken from children being treated for ALL at our unit on the UK MRC UKALL2003 trial or ALLR3 relapse trial. Samples were analyzed by 4-color flow cytometry using a large panel of monoclonal antibodies to identify all leukemia-associated immunophenotypes (LAIP). For follow up samples a minimum of 2 markers were identified for each patient and sequential gating was used for analysis. 500 000 to 106cells were acquired for each antibody combination for analysis to be sufficiently sensitive (0.01%). The number of residual leukemic cells was calculated as the percentage of blasts present within total nucleated cells counted. Sensitivity experiments were performed using dilutions of leukemic blasts in normal bone marrow or peripheral blood and demonstrated a sensitivity of 0.01%.Fifty paired BM and PB samples from 46 patients (41 newly diagnosed, 5 relapse) were included in the study (Age 2–22 years, mean 7.8 years). Of these, 22 were taken at D8 and 28 at D15. Forty-two samples were from patients with B-cell ALL, whilst 8 were from patients with T-cell ALL. The most frequent LAIPs used to identify blasts in B-ALL were CD58 (90% of cases), CD45 (75%), CD38 (86%), CD123 (77%). In T-ALL the most common LAIP combinations were CD34 (75%), CD5/CD56 (62.5%), CD5/CD2 (50%) and CD5/CD99 (50%).In keeping with previous reports, B-ALL MRD levels in PB blood were approximately 1.3-log lower than BM MRD levels (D8 mean 2.27% vs. 27.8%; D15 levels 0.63% vs. 7.99%). T-ALL levels were approximately 0.5-log lower. Importantly, the level of MRD in BM and PB in B-ALL showed a strong correlation (r=0.77).Whilst there were insufficient relapses to allow correlation with long-term outcome, PB MRD levels at D8 showed good prediction of post-induction molecular MRD, the current time point used for risk stratification in the UK trial. In patients with PB MRD >1% at D8, 100% (5/5) were in the high-risk group post-induction, significantly more than those with PB MRD <1% at D8, of which only 36.4% (4/11) were allocated to the high risk group post-induction (p=0.023). MRD levels in BM at D8 did not show similar predictive value. Importantly, this early prediction of risk could offer an early opportunity to intensify treatment in this high-risk group, potentially enhancing disease clearance during the induction phase.This study further supports that early assessment of MRD using flow cytometric techniques enhances treatment stratification in pediatric ALL. Furthermore, it shows that PB sampling may provide a markedly less invasive means of MRD assessment in these patients. Validation in a larger cohort of patients is warrented. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin Induced Erythroid Precursor Pool Depletion Causes Erythropoietin Hyporesponsiveness

Abstract 5156 Background:Erythropoietin (EPO) hyporesponsiveness is demonstrated by a persistence of anemia despite high dose of recombinant human erythropoietin (rHuEPO) or requirement of large doses to maintain the target hemoglobin concentration. The purpose of this study is to demonstrate that the bone marrow depletion induced by rHuEPO treatment may another contributing factor to hyporesponsiveness. Methods:Healthy Wistar rats were given single dose (SD) or multiple doses (MD) of rHuEPO. In MD study, animals were challenged with thrice-weekly (100 IU/kg) over two weeks and the pharamcodynamic biomarkers included reticulocyte (RET) and red blood cells (RBC) counts, and hemoglobin (HGB) concentrations. In SD study, in addition to the biomarkers in the peripheral blood, the erythropoietic responses in bone marrow and spleen were also quantified using a flow cytometric immunophenotyping technique. The total marrow and spleen cellularity was quantitatively assessed using flow cytometry. A mathematical approach involving measuring RET age distribution was developed to evaluate the RET loss due to neocytolysis. The pharmacokinetics of rHuEPO was assessed by measuring serum concentrations over time using ELISA. Result:In MD study, a slow and oscillatory decline of RET response was observed before the administration of the last dose on day 11. The RET kept decreasing below the baseline and reached a nadir on day 22, followed by a slow return to the baseline on day 28. These observations demonstrated the tolerance and rebound phenomena. In SD study, the RET decreased below the baseline after day 6. A depletion of the bone marrow erythroid precursor cells was also observed. Meanwhile, neocytolysis of RET was only observed from day 3–5 after rHuEPO injection. These results suggested that the reduced levels of RET after day 6 resulted from the depletion in the erythroid precursor cells in bone marrow induced by rHuEPO treatment. Conclusion:The EPO hyporesponsiveness is of clinical importance because it is associated with an increased risk of death or cardiovascular events. The findings in this study demonstrate that EPO-induced erythroid precursor depletion may contribute to the rHuEPO hyporesponsiveness. When the erythroid precursor cells are depleted, an increase of rHuEPO dose would not show much benefit, due to the smaller number of cells present to initiate the erythropoiesis. These findings provide additional justification for reducing the doses of erythropoietin stimulating agents in anemic patients demonstrating hyporesponsiveness. Disclosures:No relevant conflicts of interest to declare.

Characterization of functionally distinct mitochondrial subpopulations

Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca(2+) concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry. We validated that side scatter was a better indicator of volume and that as side scatter (SSC) decreased mitochondrial volume increased. Furthermore, those mitochondria with the highest NAO content had greater side scattering and were most highly charged. Mitochondria with average NAO content were of average side scattering and maintained an intermediate charge. Those mitochondria with low NAO content had minimal side scattering and exhibited minimal charge. Upon titration with the uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was found that the high NAO content subpopulations were more resistant to uncoupling than lower NAO content populations. Ca(2+)-induced swelling of mitochondria was evaluated using probability binning (PB) analyses of SSC. Interestingly, only 30% of the mitochondria showed changes in response to Ca(2+), which was blocked by cyclosporine A. In addition, the small, high NAO content mitochondria swelled differentially in response to Ca(2+) over time. Our results demonstrate that flow cytometry can be used to identify mitochondrial subpopulations based on high, mid and low NAO content and/or volume/complexity. These subpopulations showed differences in membrane potential, volume, and responses to uncoupling and Ca(2+)-induced swelling.

EFFECT OF CIGARETTE SMOKE EXTRACT ON THE EXPRESSION OF THROMBOMODULIN IN HUMAN ENDOTHELIAL CELLS

ObjectivesCigarette smoking cause triggering of coronary thrombosis. However, the mechanism by which smoking cause arterial thrombosis remains unknown. Thrombomodulin (TM) is constitutively expressed on the endothelial cell surface. TM has...

Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Stimulated by Hypoxia Promote Angiogenesis Both In Vitro and In Vivo

Although mesenchymal stem cells (MSCs) have been increasingly trialed to treat a variety of diseases, the underlying mechanisms remain still elusive. In this study, human umbilical cord (UC)-derived MSCs were stimulated by hypoxia, and the membrane microvesicles (MVs) in the supernatants were collected by ultracentrifugation, observed under an electron microscope, and the origin was identified with the flow cytometric technique. The results showed that upon hypoxic stimulus, MSCs released a large quantity of MVs of ~100 nm in diameter. The MVs were phenotypically similar to the parent MSCs, except that the majority of them were negative for the receptor of platelet-derived growth factor. DiI-labeling assay revealed that MSC-MVs could be internalized into human UC endothelial cells (UC-ECs) within 8 h after they were added into the culture medium. Carboxyfluorescein succinimidyl ester-labeling technique and MTT test showed that MSC-MVs promoted the proliferation of UC-ECs in a dose-dependent manner. Further, MVs could enhance in vitro capillary network formation of UC-ECs in a Matrigel matrix. In a rat hindlimb ischemia model, both MSCs and MSC-MVs were shown to improve significantly the blood flow recovery compared with the control medium (P<0.0001), as assessed by laser Doppler imaging analysis. These data indicate that MV releasing is one of the major mechanisms underlying the effectiveness of MSC therapy by promoting angiogenesis.

Effect of &lt;i&gt;Allium cepa&lt;/i&gt; and &lt;i&gt;Allium sativum&lt;/i&gt; on some immunological cells in rats

Extracts of some spices have been reported to play a contributory role in enhancing immune function. We evaluated and compared the effect(s) of single and combined oral administration of fresh aqueous onion (Allium cepa) and garlic (Allium sativum) extracts at different concentrations on some immunological determinants in rats. CD₄ cells of the rats were estimated using Partec flow cytometric technique, while total and differential white blood cell (WBC) counts were estimated using the Sysmsex® automated haematology analyzing technique. Our findings revealed that, CD4 and total WBC counts were significantly increased (P≤0.05) in a dose-dependent manner in both onion (250mg/Kg/d: 349±11cell/ul and 2.75±0.15X10³cell/l; 500mg/Kg/d: 389±10cells/µl and 3.05±0.05 X10³cell/l; 750mg/Kg/d: 600±11cell/µl and 3.25±0.05X10³cells/l) and garlic (250mg/Kg/d: 410±10cell/ul and 2.85±0.15X10³cell/l; 500mg/Kg/d: 494±32cells/µl and 3.30±0.10 X10³cell/l; 750mg/Kg/d: 684±11cell/µl and 3.55±0.05X10³cells/l) treated rats when compared to the zero control (200±11cells/µl and 1.55±0.05X10³cells/l, respectively). Extract of garlic at 750mg/Kg/d had significantly increased the CD4 cells and total white cell count when compared to other concentrations (P≤0.05). However, no significant effect was observed on these parameters when extracts were combined (250mg/Kg/d: 252±21cell/µl and 1.80±0.10X10³cells/l; 500mg/Kg/d: 315±21cells/ul and 2.10±0.10X10³cells/l; 750mg/Kg/d: 368±10cells/µl and 2.35±0.05X10³cells/l, respectively), the differential WBC count showed a significant increase in the proportion of cell types (lymphocytes, neutophils and monocytes) (P≤0.05). The results from this study revealed the immune boosting capabilities of Allium cepa and Allium sativum, but underscored their synergistic activities.

Picoplankton Community Composition by CARD-FISH and Flow Cytometric Techniques: A Preliminary Study in Central Adriatic Sea Water

Data concerning picoplanktonic community composition and abundance in the Central Adriatic Sea are presented in an effort to improve the knowledge of bacterioplankton and autotrophic picoplankton and their seasonal changes. Flow cytometry analyses revealed the presence of two distinct bacteria populations: HNA and LNA cells. HNA cells showed an explicit correlation with viable and actively respiring cells. The study of viability and activity may increase our knowledge of the part that contributes really to the remineralization and bacterial biomass production. Authotrophic picoplankton abundance, especially picocyanobacteria, was strongly influenced by seasonality, indicating that light availability and water temperature are very important regulating factors. In terms of total carbon biomass, the main contribution came from heterotrophic bacteria with a lower contribution from autotrophic picoplankton. CARD-FISH evidenced, within the Eubacteria domain, the dominance of members of the phyla Alphaproteobacteria, with a strong contribution from SAR11clade, followed by Cytophaga-Flavobacterium and Gammaproteobacteria. The bacterial groups detected contributed differently depending when the sample was taken, suggesting possible seasonal patterns. This study documents for the first time picoplankton community composition in the Central Adriatic Sea using two different approaches, FCM and CARD-FISH, and could provide preliminary data for future studies.

CTLA4Ig Prevents Alloantibody Formation Following Nonhuman Primate Islet Transplantation Using the CD40-Specific Antibody 3A8

Islet transplantation to treat type 1 diabetes has been limited in part by toxicities of current immunosuppression and recipient humoral sensitization. Blockade of the CD28/CD80/86 and CD40/CD154 pathways has shown promise to remedy both these limitations, but translation has been hampered by difficulties in translating CD154-directed therapies. Prior CD40-directed regimens have led to prolonged islet survival, but fail to prevent humoral allosensitization. We therefore evaluated the addition of CTLA4Ig to a CD40 blockade-based regimen in nonhuman primate (NHP) alloislet transplantation. Diabetic rhesus macaques were transplanted allogeneic islets using the CD40-specific antibody 3A8, basiliximab induction, and sirolimus with or without CTLA4Ig maintenance therapy. Allograft survival was determined by fasting blood glucose levels and flow cytometric techniques were used to test for donor-specific antibody (DSA) formation. CTLA4Ig plus 3A8, basiliximab and sirolimus was well tolerated and induced long-term islet allograft survival. The addition of CTLA4Ig prevented DSA formation, but did not facilitate withdrawal of the 3A8-based regimen. Thus, CTLA4Ig combines with a CD40-specific regimen to prevent DSA formation in NHPs, and offers a potentially translatable calcineurin inhibitor-free protocol inclusive of a single investigational agent for use in clinical islet transplantation without relying upon CD154 blockade.

In vivo flow cytometric Pig‐a and micronucleus assays: Highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated‐dose designs

Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood-based genotoxicity endpoints-micronucleated reticulocyte frequency and gene mutation at the Pig-a locus-thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short-term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague-Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3-day and 28-day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig-a mutation with a dual labeling method (SYTO 13 in combination with anti-CD59-PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3-day study (43-fold increase) and on Day 42 of the 28-day study (171-fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28-day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate that Pig-a and micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that the Pig-a endpoint is suitable for both repeated-dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on-going toxicology studies, and use as a short-term assay that provides efficient screening and mechanistic information in vivo.

Inhibition of HIV-1 tat-induced transactivation and apoptosis by the divalent metal chelators, fusaric acid and picolinic acid—Implications for HIV-1 dementia

The HIV-1 transactivator protein tat is pivotal to the pathogenesis of AIDS, exerting its effects on both viral and cellular gene expression. The basic structure of tat protein allows it to be secreted by HIV-1 infected cells and penetrate uninfected cells where it elicits its multifunctional biochemical effects. The main function of tat protein is viral transactivation which leads to the upregulation of transcription through complex interactions with RNA and host cell factors. Since HIV-1 has been widely implicated as a causative agent of HIV-1 dementia, the aim of our study was to investigate the ability of two novel metal chelators, fusaric acid (FA) and picolinic acid (PA) to firstly inhibit HIV-1 tat induced transcription and secondly, to minimize its cytotoxic effects as mediated via apoptosis. Biologically active tat protein is not freely available commercially. We therefore had to produce, isolate and purify our own protein. A cell culture system and flow cytometric techniques were used in our study. Exposure of CEM-GFP cells to exogenous recombinant tat protein induced transcription and apoptosis, and both processes were inhibited by FA and PA at concentrations that alone did not induce any cytotoxicity. Our data suggest that FA and PA may have therapeutic potential in the management of HIV-1 dementia.

A comparison of the cellular actions of polaprezinc (zinc-l-carnosine) and ZnCl2

AimsZinc supplementation has been proven to be beneficial for the prevention of some health problems. Many zinc supplements are used for medical and nutritional purposes. However, it is difficult to distinguish between them in terms of their cellular actions. We compared the cellular actions of polaprezinc (zinc-l-carnosine) with those of ZnCl2 in order to determine whether polaprezinc has greater zinc-related actions than ZnCl2. Main methodsCellular actions of polaprezinc and ZnCl2 were estimated by flow-cytometric techniques with appropriate fluorescent probes in rat thymocytes. Key findingsBoth agents had almost equal stimulatory effects on the intracellular Zn2+ level and cellular level of nonprotein thiol in a similar concentration-dependent manner. However, the increase in cell lethality caused by ZnCl2 under severe oxidative stress was significantly greater than that caused by polaprezinc. SignificanceThere are various zinc supplements, for example, zinc gluconate, zinc picolinate, and zinc methionine. However, the differences in their cellular actions have not been elucidated to date. Such studies could distinguish between zinc supplements.

Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells

The magnitude and functional phenotype (e.g. proliferation, immune stimulation) of vaccine-induced T-cell responses are likely to be critical in defining responses that can control pathogenic challenge. Current multi-parameter flow cytometric techniques may not be sufficient to measure all of these different functions, since characterizing T-cell responses by flow cytometry is presently limited to concurrent measurement of at most 10 cytokines/chemokines. Here, we describe extensive studies conducted using standardized GCLP procedures to optimize and qualitatively/quantitatively qualify a multiplex bead array (MBA) performed on supernatant collected from stimulated peripheral blood mononuclear cells (PBMC) to assess 12 cytokines and chemokines of interest. Our optimized MBA shows good precision (intra-assay, inter-day, inter-technician; coefficients of variation <30%) and linearity for most of the analytes studied. We also developed positivity criteria that allow us to define a response as positive or negative with a high degree of confidence. In conclusion, we provide a detailed description of the qualification of an MBA, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. This assay provides an excellent complement to the existing repertoire of assays for assessing immunogenicity in HIV vaccine clinical trials.

Hydrodynamic stretching of single cells for large population mechanical phenotyping

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

Decreased procoagulant phospholipids in patients treated by vitamin K antagonists

IntroductionThe stimulation of cells by thrombin is associated with the release of microparticles (MPs) from cell membranes. These MPs can express procoagulant activity. As vitamin K antagonists (VKA) decrease the generation of thrombin, we compared plasma procoagulant phospholipids (PPL) levels in patients with a previous history of venous thrombosis who were being treated with VKA and compared them with an untreated group. Materials and methodsPlasma PPL were measured using a factor Xa-based coagulation assay. sGPV, a marker of platelet activation by thrombin, was measured by ELISA. Platelet MPs were also evaluated using standard flow cytometric techniques. Ninety-six VKA-treated patients and 80 patients not undergoing VKA therapy were tested and the results compared. ResultsPPL activity was significantly reduced (p<0.0001) in VKA-treated patients compared with the untreated group. PPL were correlated with platelet and white blood cell count and with sGPV levels in the untreated group, but not in VKA-treated patients. PPL were correlated with fibrinogen levels in both groups, but not with C-reactive protein. Polymorphonuclear neutrophils (PMN) were significantly lower (p=0.01) in VKA-treated patients than in untreated patients. ConclusionThe difference between PPL levels in VKA-treated patients and patients without treatment could be related to the decrease in PMN count. It remains to be established if this decrease of PPL is directly related to the capacity of activated PMN to generate MPs, or indirectly by reducing the amount of pro-inflammatory cytokines or reactive oxygen species produced by PMN.

Prenatal Diagnosis of LAD-I on Cord Blood by Flowcytometry

To optimize a simple flowcytometric technique for Prenatal diagnosis (PND) for Leukocyte adhesions defect (LAD-I) on cordocentesis sample at 18wk gestation. Normal reference ranges for expression of CD18/CD11-integrins in neutrophils and lymphocytes at 18wk of gestation were established by flowcytometry. PND for LAD-I was then performed on the cordocentesis samples in three 'at risk' pregnancies after ruling out maternal contamination. CD18 and CD11a expression on fetal lymphocytes were found to be the most useful parameters for PND of LAD-I. All the three fetuses tested showed normal expression of CD18/CD11-integrins and thus were unaffected. This was confirmed by testing the cord blood (CB) samples after delivery and normal growth and absence of serious infections on follow-up. Flowcytometry offers a rapid and sensitive technique for PND of LAD-I in the absence of facilities for molecular diagnosis. Obstetricians, even in developing countries with modest facilities, can offer considerable relief for the families.

Changes of Treg-Associated Molecules on CD4+CD25+Treg Cells in Myasthenia Gravis and Effects of Immunosuppressants

Myasthenia gravis (MG) is a CD4(+) T cell-dependent autoimmune disease, and close attention has been paid to the role of CD4(+)CD25(+)Treg cells (Tregs). Previous results regarding Tregs in MG patients have been conflicting. The discrepancy was partly ascribed to selecting different Treg-associated molecules in defining Tregs. Therefore, we considered it necessary to find a reliable index for assessing the immunologic state in MG patients and explore the effect of IS on them. We adopted flow cytometric techniques to measure the numbers and frequencies of Tregs in peripheral blood taken from 57 patients and 91 age-matched healthy donors, and we also analyzed FOXP3 mean fluorescence intensity on Tregs. The number and frequency of Tregs in peripheral blood of MG patients significantly decreased, together with down-regulation of FOXP3 expression. There was dynamic change of Treg cell level and the inverse relationship with clinical symptom, suggesting that the immunologic disorder in MG patients was related to peripheral Tregs population. Meanwhile, CD4(+)CD25(+)FOXP3(+)Helios(+)T cells might be activated Tregs, rather than nTregs. Moreover, the number and frequency of CD4(+)CD25(+)FOXP3(+)Helios(+)T cells significantly decreased in MG patients, indicating that the reduction of the activated Tregs population might be a critical contributor to the pathogenesis of MG. The significant reduction of the peripheral Tregs population in MG patients might be responsible for the immunologic disorders in MG patients. IS such as GC took its effect possible by increasing the population size, and the underlying mechanism should be further investigated.