Cytometric Bead-based Assay Research Articles

Microvascular activation and exocytosis after exposure to the serum from mismatched recipients by using donor microvascular cultures

BackgroundMicrovascular injury resulting from activation and exocytosis are early signs of tissue damage caused by allografting. However, humoral anti-graft reactions are not easily detectable in transplant biopsies. The aim of this study was to establish a bioassay to recapitulate this process in a prospective approach. MethodsThe study was executed by using our previously established protocol to isolate and freeze the donors' microvascular endothelial cells (MVEC) at the transplantation (34 living-related donors and 26 cadaver donors); and to collect sera from the recipients before the transplantation, one-, three- and six-months after transplantation. The activation and exocytosis of the MVEC were determined by incubating the donors' cultures with the recipients' sera. We determined if there was any endothelial activation by quantifying the releases of monocyte chemotactic protein-1 (MCP-1) and interleukin 8 (IL-8) in supernatants and the expressions of membrane intercellular adhesion molecule-1 (CD54) and intercellular adhesion molecule-1 (CD106) by flow cytometry. Endothelial exocytosis was determined by quantifying soluble E-selectin (CD62E) and cytoplasmic von Willebrand Factor (vWF) in supernatants. Endothelial activation or exocytosis was considered positive when the fold change (≧1.5) of post-transplantation to pre-transplantation was reached. We also monitored serum PRA and cytokines using Luminex multiple-plex and cytometric bead-based assay respectively. ResultsWe found 41.2% recipients (14 out of 34, ranging from 1.5 to 5.2 folds, p < 0.05) exhibited positive MVEC activation in the first month after transplantation as determined by IL-8 levels; 26.5% recipients (9 out of 34, ranging from 1.5 to 11.8 folds, p < 0.05) by MCP-1 levels. In the group of three months post-transplantation, 70.6% patients were positive (12 out of 17, ranging from 1.8 to 87.1 folds, p < 0.05) by IL-8 increased levels; 24% recipients (4 out of 17, ranging from 1.8 to 50.5 folds, p < 0.05) measured by MCP-1 levels. However, these changes disappeared six months after transplantation. Flow cytometric data showed that a time-dependent of CD54+ and CD106+ expressions existed over the course of six months. Most CD54+ and CD106+ cells were CD31- (platelet-endothelial cell adhesion molecule-1), though CD31+/CD106+ (37.5%, 3 out of 8) and CD31+/CD106+ (25%. 2 out of 8) were seen. When comparing donor MVEC activation to their recipient's proinflammatory cytokine levels or PRA status, we could not draw a conclusion regarding the connections between them. The sera collected from recipients at either one- or three-months after allografting did not significantly induce the release of either soluble CD62E or vWF (p > 0.05), indicating exocytosis was not significantly involved in the acute phase of allografting. ConclusionsThis bioassay enables us to detect the activation and exocytosis of donor MVEC elicited by respective sera from mismatched kidney recipients.

The role of vascular inflammation markers in deficiency of adenosine deaminase 2

ObjectivesThe first objective was to assess the role of vascular inflammatory factors in the pathogenesis of deficiency of adenosine deaminase 2 (DADA2) and to compare these markers among DADA2 patients with different phenotypes. We also aimed to investigate differences between DADA2 patients with vasculitic features and classic polyarteritis nodosa (PAN) for the aforementioned markers. MethodsThe study included eighteen DADA2 patients, ten PAN patients, and eight healthy controls. Plasma levels of sST2, sRAGE, Tie-2, sCD40L, Tie-1, sFlt-1, LIGHT, TNF-α, PlGF, IL-6, IL-18, IL-10, MCP-1 were studied by cytometric bead-based multiplex assay panel. ResultsAmong the DADA2 patients, five had hematological manifestations, 13 had vasculitic findings, and accompanying immunological findings were present in seven patients. Nine patients had neurological findings, five of whom had neuropathy. Plasma levels of Tie-1 and sFlt-1 were higher in the overall DADA2 patients compared to healthy controls and PAN patients (p<0.001 and p = 0.004, respectively). DADA2 patients with PAN-like features had higher sRAGE, Tie-2, and TNF-α levels compared to PAN patients (p = 0.013, p = 0.003, and p = 0.001, respectively).In DADA2 patients with hematological findings, plasma IL-18 levels were higher than those with PAN-like phenotype (p = 0.001). Finally, DADA2 patients with neuropathy had higher sRAGE concentrations than patients without neuropathy and healthy controls (p = 0.03 and p = 0.008, respectively). ConclusionsWe suggest that the high plasma IL-18 levels observed in DADA2 patients with hematologic manifestations may be associated with an activated IFNγ pathway, and lack of response to anti-TNF treatment. We identified sRAGE as a potential biomarker of neuropathy in DADA2 patients. Higher concentrations of Tie-1, Tie-2, sFlt-1, sRAGE, and TNF-α distinguished DADA2 patients with PAN-like features from PAN patients.

Plasma checkpoint protein levels and galectin-9 in juvenile systemic lupus erythematosus.

SLE is a disease of the adaptive immune system where T lymphocyte dysfunction has an important role as well. We assessed the plasma levels of checkpoint receptors expressed on T cells, along with Galectin-9 to reflect type-1 IFN activity and IL-2Rα in childhood SLE patients. Forty-nine children with SLE and15 healthy controls were included. SLEDAI scores were evaluated at the time of sampling. CD25 (IL-2Rα), 4-1BB, B7.2 (CD86), TGF-β1, CTLA-4, PD-L1, PD-1, Tim-3, LAG- 3, Galectin-9 levels were studied by cytometric bead-based multiplex assay panel. Galectin-9 and PD-L1 were significantly higher in SLE patients. Other checkpoint proteins and IL-2Rα were also higher but did not reach statistical significance. There were significant correlations between SLEDAI and IL-2Rα, Galectin-9 and PDL1. There were three clinical clusters: Cluster 1 included patients with no major organ involvement, cluster 2 had predominantly haematological involvement(n=16) and cluster 3 (n=11) had predominantly renal involvement. Checkpoint proteins were not different among these three clusters. Our data supports that Galectin 9 and IL-2Rα are good markers for disease activity in childhood SLE. We need larger series to evaluate differences between disease clusters in SLE. We failed to show a significant correlation with checkpoint proteins and SLEDAI except for PDL1.

Activated low-density granulocytes in peripheral and intervillous blood and neutrophil inflammation in placentas from SLE pregnancies

ObjectiveWomen with SLE face an increased risk of adverse pregnancy outcomes compared with healthy women, but the underlying immunological mechanisms are unknown. Given the recognised association of neutrophil activation with...

Regulation of cytokine production by treating with Chinese tonic herbs in human peripheral blood mononuclear and human acute monocytic leukemia cells

Chinese tonic herbs are often used as remedies for the treatment of cancer or immunity related diseases. Cytokine regulation is one of the pathways that modulates the immune system. In the present study, we investigated the effects of commonly used Chinese herbs on cytokine regulation in human peripheral blood mononuclear cells (PBMC) and human acute monocytic leukemia cell line, THP-1. Among the 15 selected Chinese herbs, Paeonia lactiflora exhibited inhibitory effect on phytohemagglutinin (PHA)-activated PBMC proliferation and induced IFN-γ production. We also used a multiplexed flow cytometric bead-based assay to analyze the release of 17 cytokines and reverse transcription polymerase chain reaction (RT-PCR)/quantitative real-time PCR (qRT-PCR) to evaluate gene expression. Cuscuta chinensis induced both interleukin-8 and MIP-1β release in THP-1 cells and influenced mRNA transcription. Dioscorea opposita, Cinnamomum osmophloeum, and Glycyrrhiza uralensis enhanced secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) and influenced GM-CSF gene expression. As such, different Chinese tonic herbs may regulate immunomodulatory functions in human cells via diverse processes such as the modulation of different cytokines and may be used as adjuvant immunity-boosting drugs. The present study is the first to use high-throughput multiplexed flow cytometric method to analyze cytokine regulatory effects of herbal extracts.

Evaluation of the Immunoprotective Potential of Recombinant Paraflagellar Rod Proteins of Trypanosoma evansi in Mice.

Trypanosomosis, caused by Trypanosoma evansi, is an economically significant disease of livestock. Systematic antigenic variation by the parasite has undermined prospects for the development of a protective vaccine that targets the immunodominant surface antigens, encouraging exploration of alternatives. The paraflagellar rod (PFR), constituent proteins of the flagellum, are prominent non-variable vaccine candidates for T. evansi owing to their strategic location. Two major PFR constituent proteins, PFR1 (1770bp) and PFR2 (1800bp), were expressed using Escherichia coli. Swiss albino mice were immunized with the purified recombinant TePFR1 (89KDa) and TePFR2 (88KDa) proteins, as well as with the mix of the combined proteins at equimolar concentrations, and subsequently challenged with virulent T. evansi. The PFR-specific humoral response was assessed by ELISA. Cytometric bead-based assay was used to measure the cytokine response and flow cytometry for quantification of the cytokines. The recombinant TePFR proteins induced specific humoral responses in mice, including IgG1 followed by IgG2a and IgG2b. A balanced cytokine response induced by rTePFR 1 and 2 protein vaccination associated with extended survival and improved control of parasitemia following lethal challenge. The observation confirms the immunoprophylactic potential of the covert antigens of T. evansi.

Rosuvastatin Attenuates High-Salt and Cholesterol Diet Induced Neuroinflammation and Cognitive Impairment via Preventing Nuclear Factor KappaB Pathway

Recent attention is focused on the impact of diet on health and mental well-being. High-salt and cholesterol diet (HSCD) is known to be associated with neuroinflammation which is the predominant factor for neurodegenerative disease like Alzheimer disease (AD). In the present study, we examined the neuroprotective potential of rosuvastatin, an HMG-CoA reductase inhibitor against HSCD induced neuroinflammation and cognitive impairment. Our results demonstrated that HSCD-induced cognitive impairment as determined by Morris water maze (MWM) task. HSCD also activated nuclear factor kappaB (NF-kB) signaling pathway. The cytokine response was measured using a cytometric bead-based assay quantified by flow cytometry. Treatment with rosuvastatin decreased the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and increased interleukin-10 (IL-10) in a dose-dependent manner. Our results also demonstrated that the rosuvastatin modulates neuronal cell death by inhibiting the overexpression of NF-kB in the CA1 region of hippocampus. In addition, molecular docking study of rosuvastatin indicated high affinity and tighter binding capacity for the active site of the NF-kB. These results suggest that HSCD-triggered inflammatory response and cognitive impairment may be associated with NF-κB signaling pathway. Therefore, treatment with rosuvastatin could be a potential new therapeutic strategy for sporadic dementia of AD.

Comparison of lipopolysaccharides and soluble CD14 measurement between clinically endotoxaemic and nonendotoxaemic horses.

Clinically useful biomarkers are needed for early identification of endotoxaemic horses. Soluble CD14 (sCD14) is amplified early in response to inflammatory signals, including bacterial lipopolysaccharide (LPS), and may prove a useful biomarker for clinical endotoxaemia. The aim of this study was to determine if sCD14 could serve as a more reliable biomarker of the clinical signs of endotoxaemia, compared to measuring LPS alone. Prospective observational study in horses at a veterinary teaching hospital. Plasma samples were collected from 20 healthy horses and 35 horses presenting for emergency evaluation. Horses were classified as clinically endotoxaemic, using previously established criteria, if they had a heart rate >70 beats/min, packed cell volume >45% and/or a lesion likely to result in endotoxaemia. Soluble CD14 was measured using a cytometric bead-based assay and LPS was measured using a Limulus amoebocyte lysate (LAL) assay. Soluble CD14 was higher in horses classified as clinically endotoxaemic (median 1102 ng/ml, interquartile range 439 ng/ml), compared to clinically nonendotoxaemic (median 692 ng/ml, interquartile range 455 ng/ml, P = 0.03. There was no difference in LPS concentrations between clinically nonendotoxaemic (median 5.4 endotoxin units [EU]/ml, interquartile range 5 EU/ml) and endotoxaemic horses (median 7.2 EU/ml, interquartile range 17 EU/ml, P = 0.2). There was no correlation between sCD14 and LPS values in paired serum samples. LPS and sCD14 values were used to generate a receiver operating characteristic curve. The area under the curve for LPS and sCD14 was <0.7, suggesting that sCD14 and LPS were poor predictors of clinical endotoxaemia for the horses in this study. Further investigation is warranted to assess the utility of sCD14 measurement as a clinically useful biomarker to identify endotoxaemia in horses.

Protective antibody and cytokine responses in mice following immunization with recombinant beta-tubulin and subsequent Trypanosoma evansi challenge.

BackgroundTrypanosomosis or Surra, caused by the flagellated hemoprotozoan parasite Trypanosoma evansi, is a disease of economic importance through its wide prevalence in domestic livestock in tropical countries. In the absence of a protective vaccine, management of the disease relies on a few available chemotherapeutic agents. Although humoral immunity is the mainstay of resistance to T. evansi, the ability of the parasite to vary its immunodominant surface proteins to subvert the immune system has forced vaccine efforts to target a variety of invariant epitopes. Beta tubulin, an integral component of the trypanosome cytoskeleton, was therefore targeted using the recombinant form of the protein for immunization.MethodsThe 1329 bp coding sequence of beta tubulin gene was PCR amplified and cloned in pQE-TriSystem expression vector. Recombinant beta tubulin was heterologously expressed in Escherichia coli as a 46 KDa fusion protein and used for immunization of mice. The Ig response was studied by ELISA, whereas the cytokine response was measured using a cytometric bead-based assay quantified by flow cytometry.ResultImmunization with recombinant beta (β)-tubulin protein induced a beta-tubulin specific humoral immune response of predominantly IgG2a isotype. Lethal challenge with T. evansi blood-form trypomastigotes post-immunization elicited a robust anamnestic response. An abundance of IFN-γ further confirmed the Th-1 bias of the protective response. We also observed extended survival and better control of the challenge infection in the immunized mice.ConclusionsA robust anamnestic response following challenge including a Th-1 serum cytokine profile coupled with increased survival is indicative of protective immunity in the immunized mice. These observations suggest that β-tubulin of T. evansi is a viable antigenic target for development of a vaccine against this important livestock pathogen.

Circulating serum sVCAM-1 concentration in advanced ovarian cancer patients: correlation with concentration in ascites

BackgroundVascular cell adhesion molecule-1 (VCAM-1) is associated with ovarian cancer progression but the origin of its soluble form (sVCAM-1) in serum is not well investigated. The purpose of this study was to elucidate whether the concentration of sVCAM-1 in serum correlates with the concentration in ascites, that represents local tumour environment, and with systemic inflammation, various clinicopathological characteristics, and patient outcome.Patients and methods.Thirty-six patients with advanced ovarian cancer were included in the study. Serum for sVCAM-1 analysis was obtained prior to surgery. Ascites samples were collected at the beginning of the operation. Clinical data were collected from patients’ medical records. sVCAM-1 in samples was analysed by flow cytometric bead-based assay. The mean follow-up period was 11 months (range 0–23) from the time of surgery.ResultsSerum sVCAM-1 concentrations are positively correlated to ascites sVCAM-1 concentrations. There was a weakly positive correlation of serum sVCAM-1 with tumour size and no correlation with inflammatory tumour markers, FIGO stage or grade. Higher concentrations of sVCAM-1 were associated with poor disease outcome (death from ovarian cancer) in almost all cases before chemotherapy was started.ConclusionsThis is the first study demonstrating that serum concentrations of sVCAM-1 in advanced ovarian cancer patients correlate with sVCAM-1 concentrations in ascites, thus expressing the biologic potential of malignant disease to metastasis, rather than systemic inflammation. Higher serum and ascites sVCAM-1 concentrations might have predictive potential for different biologic behaviour.

Prevention Of Murine FVIII-Specific Memory B Cells Due To FcγR2B (CD32) Blockade Is Associated With Distinctive Alterations In The FVIII-Specific Cytokine Profile

Development of neutralizing antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in hemophilia A. Long-term application of high doses of FVIII can induce tolerance in the context of immune tolerance therapy (ITT). Very high concentrations of FVIII have been shown to prevent the development of FVIII-specific antibody-secreting cells (ASCs) from memory B cells by inducing apoptosis. We have previously demonstrated that ASC differentiation from memory B cells is also abolished when CD32, an inhibitory Fc-gamma receptor expressed on B cells and dendritic cells, was genetically deleted or blocked by monoclonal antibodies (mAb). Here, we addressed the question how CD32 inhibition prevented ASC development by studying the FVIII-specific T cell response in the absence or presence of CD32 inhibition. Hemophilia A mice (B6;129S4-F8tmKaz/J) were immunized with recombinant human FVIII (rhFVIII) for 4 weeks, and spleen cells were re-stimulated with rhFVIII (0, 0.5, 1 or 10 IU/ml) ex vivo in the presence or absence of anti-CD16/CD32 antibody (2.4G2) to inhibit CD32. Formation of FVIII-specific ASCs was assessed on day 6 by ELISPOT. IFN-γ, IL-2, IL-4, IL-6, IL-10, and TNF-α were detected in culture supernatant using a cytometric bead-based multiplex assay on days 1 to 6. In line with previous findings, very high doses of rhFVIII (10 IU/ml) or blockade of CD32 with mAb 2.4G2 (at high or low doses of rhFVIII) inhibited the differentiation of FVIII-specific ASCs. We observed a FVIII-dose dependent increase in the secretion of IFN-γ, IL-2, IL-4, IL-6, and IL-10. Very high doses of rhFVIII (10 IU/ml) suppressed ACS formation, but not the formation of these cytokines indicating an intact FVIII-specific T cell response. Secretion of TNF-α appeared not to be FVIII-dose dependent and was also observed in the cultures without rhFVIII. Inhibition of CD32 with mAb 2.4G2 in the presence of ASC stimulating FVIII concentrations (e.g. 1 IU/ml) prevented the development of ASC, but also diminished the formation of IFN-γ (334.2 ± 58.0 pg/ml versus 14.9 ± 1.4 pg/ml) and significantly reduced IL-10 (297.7 ± 78.5 pg/ml versus 131.3 ± 26.8 pg/ml). IL-6 was only slightly reduced, whereas IL-4 remained unchanged. IL-2 was even increased at later time points during cell culture (day 4: 16.1 ± 3.4 pg/ml versus 24.0 ± 1.4 pg/ml, day 6: 3.1 ± 0.6 pg/ml versus 21.8 ± 3.6 pg/ml). These results indicate that very high doses of FVIII prevented ASC formation, but not FVIII-specific T cell stimulation. In contrast, inhibition of CD32 prevented ASC formation, but also changed the secretion of T cell dependent cytokines. The lack of IFN-γ and IL-10 production after re-stimulation with various doses of rhFVIII indicates a reduced stimulation of Th1 and Th2 helper cells. Similar effects have been described previously, when B7-1 co-stimulation of CD4+ T cells was prevented by anti-CD80 mAb. In conclusion, inhibition of FVIII-specific ASC formation by means of very high doses of FVIII or inhibition of CD32 appears to occur differently. High doses of FVIII induce apoptosis in FVIII-specific memory B cells, but do not prevent FVIII-specific T cell responses. In contrast, inhibition of the Fcγ receptor CD32 on B cells and dendritic cells interferes with the FVIII-specific T cell response indicating a defective antigen presentation. Both high dose FVIII treatment and CD32 blockade, alone or in combination, should be further investigated to specifically address the FVIII-specific immune response in hemophilia A, and to evaluate a potential improvement of ITT. Disclosures:Vollack:Biotest AG: Research Funding. Trummer:Biotest AG: Research Funding. Tiede:CSL Behring: Consultancy, Honoraria, Research Funding; Biogen Idec: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Biotest: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Baxter: Consultancy, Honoraria, Research Funding. Werwitzke:Biotest AG: Honoraria, Research Funding.

Impact of antiretroviral therapy interruption on plasma biomarkers of cardiovascular risk and lipids: 144‐week final data from the STOPAR study

The aim of the study was to investigate changes in plasma biomarkers of cardiovascular risk and lipids in a CD4-guided antiretroviral therapy interruption study. This was a substudy of a prospective, randomized, multicentre treatment interruption study. At months 12, 24 and 36, monocyte chemotactic protein-1 (MCP-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), interleukin-6 (IL-6), interleukin-8 (IL-8), soluble CD40 ligand (sCD40L), soluble P-selectin (sP-selectin), and tissue plasminogen activator (t-PA) were measured using a multiplex cytometric bead-based assay. Total cholesterol (total-c), high-density lipoprotein cholesterol (HDL-c) and triglycerides (TG) were determined using standard methods. Fifty-four patients were included in the study [34 in the treatment continuation (TC) arm and 20 in the treatment interruption (TI) arm]. There were no differences at baseline between the groups, except in CD4 cell count, which was higher in the TI arm (P = 0.026), and MCP-1, which was higher in the TC arm (P = 0.039). MCP-1 and sVCAM-1 were increased relative to baseline at the three study time-points in the TI arm, with no changes in the TC arm. Soluble CD40L and sP-selectin were increased at month 36 in both arms, with a greater increase in the TI arm (P = 0.02). t-PA was increased in both arms at the three time-points. Total-c, HDL-c and low-density lipoprotein cholesterol (LDL-c) were decreased in the TI arm at the three time-points, with no changes in the total-c/HDL-c ratio. HIV viral load positively correlated with MCP-1 at months 12 and 24. Regression analysis showed a significant negative association of HDL-c with MCP-1 and sVCAM-1. A significant increase in cardiovascular risk biomarkers persisting over the prolonged study period was seen in the TI arm. This factor may contribute to the increased cardiovascular risk observed in previous studies.

Measurement of antibodies to pneumococcal, meningococcal and haemophilus polysaccharides, and tetanus and diphtheria toxoids using a 19-plexed assay

The measurement of antibody responses to vaccination is useful in the assessment of immune status in suspected immune deficiency. Previous reliance on enzyme-linked immunoabsorbent assays (ELISA) has been cumbersome, time-consuming and expensive. The availability of flow cytometry systems has led to the development of multiplexed assays enabling simultaneous measurement of antibodies to several antigens. We optimized a flow cytometric bead-based assay to measure IgG and IgM concentrations in serum to 19 antigens contained in groups of bacterial subunit vaccines: pneumococcal vaccines, meningococcal vaccines, Haemophilus influenzae b (Hib), and tetanus and diphtheria toxoid vaccines. 89-SF was employed as the standard serum. The assay was used to determine specific antibody levels in serum from 193 healthy adult donors. IgG and pneumococcal IgM antibody concentrations were measurable across 3 log10 ranges encompassing the threshold protective IgG antibody levels for each antigen. There was little interference between antibody measurements by the 19-plexed assay compared with monoplexed assays, and a lack of cross-reactive IgG antibody, but evidence for cross-reacting IgM antibody for 3/19 pneumococcal antigens. 90th centile values for 15/19 IgG concentrations and 12/12 IgM concentrations of the 193 adult sera were within these ranges and percentages of sera containing protective IgG antibody levels varied from 4% to 95% depending on antigen. This multiplexed assay can simultaneously measure antibody levels to 19 bacterial vaccine antigens. It is suitable for use in standard clinical practice to assess the in vivo immune response to test vaccinations and measure absolute antibody levels to these antigens.

Serum levels of interleukin (IL)-18, IL-23 and IL-17 in Chinese patients with multiple sclerosis

It has been reported that cytokines play an important role in the pathogenesis of multiple sclerosis (MS). The aim of this study was to evaluate the serum levels of interleukin (IL)-18, IL-23 and IL-17 in Chinese patients with MS. We compared the serum concentrations of pro-inflammatory cytokines IL-18, IL-23 and IL-17 in 39 patients with MS and 39 healthy controls matched with sex and age. Serum cytokines were measured by FlowCytomix, a kind of cytometric bead-based assay. Correlations between the serum levels of the three cytokines and disability (expanded disability status scale, EDSS), disease duration, current age and age at onset were examined. Serum concentrations of all IL-18, IL-23 and IL-17 were significantly higher in MS patients than healthy controls. There were no significant differences of the three cytokines' levels between female and male healthy controls, while the serum IL-18 level was observed significantly higher (P=0.049) in male MS patients than female MS patients. No significant correlations were observed between any of the three cytokines' levels and EDSS, disease duration and current age. However, IL-23 was found negatively correlated with age at onset in male MS patients (rs=−0.775, P=0.041). Our data suggest that all IL-18, IL-23 and IL-17 may be involved in the pathogenesis of MS. However, the relationships of the three cytokines and clinical characteristics of MS need to be further investigated in the future.

ACCURATE ESTIMATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR LEVELS IN MICROSAMPLES WITH A LOW-COST BEAD-BASED ASSAY

The purpose of this study was to evaluate the accuracy of measurement of a known concentration of vascular endothelial growth factor (VEGF) using cytometric bead-based assay in microsamples (50 microL) and compare it with that obtained by enzyme-linked immunosorbent assay. A known concentration of VEGF samples was prepared with addition of VEGF (100 pg/mL) to 1 mL of Hank balanced salt solution. This preparation was further diluted serially to 50, 25, and 12.5 pg/mL; and VEGF estimation was done on each of these samples using Luminex, a commercially available cytometric bead-based assay and enzyme-linked immunosorbent assay. The assay was replicated three times at each dilution. The mean concentrations of VEGF measured using Luminex were 97.7 +/- 6.2, 47.3 +/- 8.1, 24.9 +/- 2.2, and 14.2 +/- 1.8 pg/mL at 100, 50, 25, and 12.5 pg/mL dilutions, respectively. Similarly, the mean concentrations of VEGF measured using enzyme-linked immunosorbent assay for equivalent dilutions were 100.6 +/- 24.4, 53.8 +/- 16.8, 44.8 +/- 35.5, and 14.19 +/- 0.7 pg/mL, respectively. Bead-based assay accurately measures VEGF concentration in a microsample and has better validity and reliability as compared with enzyme-linked immunosorbent assay.

Diagnosing HIV infection using flow cytometry: From antigenic analyses to a specifically dedicated bead‐based assay to measure viral load

SINCE the discovery of HIV in 1984 (1,2), millions have been infected and have died of AIDS. In 2006, the World Health Organization identified 40 million HIV-infected subjects, noting that antiretroviral therapy was required throughout the world (Fig. 1; http://www.who.int/hiv/data/en/index.html). Today, the situation is very similar and the HIV pandemic remains a major world health problem (3). The treatment of AIDS with highly active antiretroviral therapy (HAART) involves regular monitoring of different blood parameters, requiring the development of convenient and accurate methods that can evaluate HIV infection even in resource-poor countries (4,5). In addition to CD4 cell count, which is described as the best surrogate marker (6–8), blood serum virus concentration (viral load) is the most meaningful parameter to evaluate treatment success and to decide on initiating HAART (9). Virus detection is also important for evaluation and prevention of mother-to-child transmission (10) and in persons with an atypical disease course (11). Viral load tests are usually performed in specialized laboratories because they require specifically dedicated materials and are based on highly specific real-time PCR, to determine viral serum concentration over a wide scale. Therefore, these tests can be difficult to conduct in developing countries. I have, therefore, read with great interest the paper by Greve et al. (12), published in the current issue of Cytometry Part A, describing a new affordable flow cytometric bead-based assay to measure HIV-1 viral load. This test, combining PCR, a novel and original bead-based assay, and flow cytometric analysis, clearly shows that various HIV1 subtypes can be identified and that the sensitivity of the method makes it possible to quantify as few as 50 copies of virus. Interestingly, the copreparation and amplification of an internal standard provides quality control of all the steps from RNA purification over PCR-reaction to the quantification of the amplified HIV-1 product. Currently, flow cytometry bead-based assays (13–15) can be carried out either on specifically dedicated machines (Luminex Technology) or on various conventional flow cytometers (16,17). They are frequently used to simultaneously identify and quantify cytokines, adipokines, growth factors, and auto-antibodies as well as cancer, cardiac, and neurological markers in a small volume (10–50 ll) of different body fluids (plasma, serum, tears, cerebrospinal fluid, synovial fluid, and bronchoalveolar lavage fluid). These methods can also be used on culture media or on cellular extracts (18). In the latter, bead-based assays are promising tools to study fusion proteins in leukemic cells (19) or protein phosphorylation on culture cells or tissue samples to investigate cell signaling under various conditions (20). The ability to combine PCR methods with cytometric bead assays was previously reported by various authors (21–23), and the method was successfully used to investigate DNA polymorphism (24,25). In virological diagnosis, detecting HIV with the combined use of PCR on a conventional flow cytometer is an ori-

Multiple Cytokine Analysis in Human Tears: An Optimized Procedure for Cytometric Bead-Based Assay

Purpose: To compare instruments and reagents used for cytometric bead-based assay (CBA) of cytokines in human tears. Methods: Two CBA instruments and reagent kits from five vendors were compared in parallel. Vendor protocols were modified to accommodate tear volumes. Precision, recovery, dilution linearity, and stability were evaluated. Results: Luminex™ and Bio-Rad™ proved optimal for tear CBA. Good correlation for dilution linearity was observed (r > 0.80, p < 0.05) for 1–4-μ l sample volumes. Samples were stable for 30 min at 25°C. Conclusion: Many tear cytokines can be reliably measured by CBA using an optimized protocol. Concentrations remain stable during periods consistent with collection.