Polystyrene microspheres enable 10-color compensation for immunophenotyping of primary human leukocytes.

Compensation is a critical process for the unbiased analysis of flow cytometry data. Numerous compensation strategies exist, including the use of bead-based products. The purpose of this study was to determine whether beads, specifically polystyrene microspheres (PSMS) compare to the use of primary leukocytes for single color based compensation when conducting polychromatic flow cytometry. To do so, we stained individual tubes of both PSMS and leukocytes with panel specific antibodies conjugated to fluorochromes corresponding to fluorescent channels FL1-FL10. We compared the matrix generated by PSMS to that generated using peripheral blood mononuclear cells (PBMC). Ideal for compensation is a sample with both a discrete negative population and a bright positive population. We demonstrate that PSMS display autofluorescence properties similar to PBMC. When comparing PSMS to PBMC for compensation PSMS yielded more evenly distributed and discrete negative and positive populations to use for compensation. We analyzed three donors' PBMC stained with our 10-color T cell subpopulation panel using compensation generated by PSMS vs.PBMC and detected no significant differences in the population distribution. Panel specific antibodies bound to PSMS represent an invaluable valid tool to generate suitable compensation matrices especially when sample material is limited and/or the sample requires analysis of dynamically modulated or rare events.

Methods of effective conjugation of antigens to nanoparticles as non-inflammatory vaccine carriers.

It has recently become clear that nanoparticle size is a major determinant for how antigen presenting cells (APCs), and specifically dendritic cells (DC) recognize and handle particles, and hence a critical parameter for the formulation of particulate vaccines that aim to induce immunity by targeting DC. Our previous studies in mice and sheep have shown polystyrene nanoparticles of 40-50 nm (PSNPs) with covalently bound antigen offer a new class of vaccines, which contain only 2 elements, antigen and particle, and no added inflammatory stimuli, but evoke very potent combined CD8 T cell and antibody responses. Herein we have optimized the methods for antigen conjugation to PSNPs to controllably promote a single antigen (protein or peptide) layer coating on the nanoparticle. Surprisingly, these nanovaccines not only continued to induce high levels of CD8 T cells in vivo, but were further more potent antibody inducers than nanoparticles containing multiple antigen layers. Addressing the issue of antigen loading on PSNPs, we found an optimal range, above or below which immunogenicity is changed either for antibodies or CD8 T cells. The mechanism behind the induction of high levels of CD8 T cells was further explored by assessing the DC subset that takes up the PSNPs in vivo, and these were found to be preferentially CD8(+) CD11c(+) DC in the lymph node draining the injection site. Since the levels of induced antibodies were highly elevated, and CD8(+) DC do not traditionally induce antibodies, we further sought to find if, despite no detectable inflammation at the injection site, the PSNPs may perhaps induce inflammatory cytokines locally in the lymph node after injection, or systemically in sera, resulting in an adjuvant effect. The initial findings presented herein show no detectable induction of the key inflammatory cytokines such as TNF-α, IL-1 or IL-6, suggesting a novel "non-inflammatory" adjuvant mechanism.

The signalling imprints of nanoparticle uptake by bone marrow derived dendritic cells.

Nanoparticles (NP) possess remarkable adjuvant and carrier capacity, therefore are used in the development of various vaccine formulations. Our previous studies demonstrated that inert non-toxic 40-50 nm polystyrene NP (PS-NP) can promote strong CD8 T cell and antibody responses to the antigen, in the absence of observable inflammatory responses. Furthermore, instillation of PS-NP inhibited the development of allergic airway inflammation by induction of an immunological imprint via modulation of dendritic cell (DC) function without inducing oxidative stress in the lungs in mice. This is in contrast to many studies which show that a variety of ambient and man-made NP promote lung immunopathology, raising concerns generally about the safe use of NPs in biomedicine. Most NPs are capable of inducing inflammatory pathways in DC largely mediated by signalling via the extracellular signal-regulated kinase 1/2 (ERK). Herein, we investigate whether PS-NPs also activate ERK in DC in vitro. Our data show that PS-NP do not induce ERK activation in two different types of bone marrow derived (BM) DC cultures (expanded with GM-CSF or with GM-CSF together with IL-4). The absence of such signalling was not due to lack of PS-NP uptake by BM-DC as confirmed by confocal microscopy and flow cytometry. The process of NP uptake by DC usually initiates ERK signalling, suggesting an unusual uptake pathway may be engaged by PS-NPs. Indeed, data herein showns that uptake of PS-NP by BM-DC was substantially inhibited by phorbol myristate acetate (PMA) but not cytochalasin D (CCD), suggesting an uptake pathway utilising caveole for PS-NP. Together these data show that BM-DC take up PS-NP via a caveole-dependent pathway which does not trigger ERK signalling which may explain their efficient uptake by DC, without the concomitant activation of conventional inflammatory pathways.

Size-dependent uptake of particles by pulmonary antigen-presenting cell populations and trafficking to regional lymph nodes.

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3⁺ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4⁺ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity.

Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca(2+). The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor κB ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 µm) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

Fabrication of anionic sulfate-functionalized nanoparticles as an immunosensor by protein immobilization.

Anionic sulfate (SO(4)(-))-functionalized polystyrene (PS) nanoparticles were prepared by the thermal decomposition of potassium persulfate (KPS) in the presence of sodium tetraborate via emulsion polymerization. The presence of a SO(4)(-) group at a solid/liquid interface of a particle surface was confirmed by a zeta potential value of -40.6 mV as well as the shifting of S 2p spectra toward a lower-binding-energy region around 162.7 eV (2p(3/2)) and 164.4 eV (2p(1/2)) in X-ray photoelectron spectroscopy (XPS) analysis. The electrostatic attraction between positively charged antibodies of human immunoglobulin G (hIgG) and cardiac troponin I (cTnI) and negatively charged particle surfaces was accomplished. The atomic force microscopy (AFM) measurement and bicinchoninic acid (BCA) assay results show binding structure between hIgG and antibodies of hIgG (anti-hIgG) with a gradual increase in particle diameter to 152.6 nm (bare), 170.2 nm (hIgG), and 178.9 nm (hIgG/anti-hIgG). Surface coverage densities of 331.4 ng/cm(2) (hIgG) and 320.3 ng/cm(2) (cTnI) and the binding capacity of hIgG to HyLite-750-labeled Fab-specific anti-hIgG (approximately 81.2%) indicate that the majority of hIgG was immobilized with a Y-shaped orientation. The sandwich immunoassay results provide the evidence that the immunological activity of cTnI on the PS nanoparticle surface was retained because the binding activity of the cTnI-PS nanoparticle/cTnI (antigen)/detection cTnI-antibody reaction showed a 5-fold higher activity than that of the cTnI-PS nanoparticle/human serum albumin (HSA)/detection cTnI antibody used as a negative control.

Particle size determines activation of the innate immune system in the lung.

Our knowledge about particle size in relation to activation of the innate immune system is limited. Therefore, the acute effect of particle exposure on the innate immune system was studied in a lung model using the intracellular bacterium Listeria monocytogenes. Female Balb/cA mice were instilled intratracheally with polystyrene particles (PSP) of different diameters (0.064, 0.202, 1.053 and 4.646 mum) simultaneously with or 1 day prior to inoculation of 10(5) bacteria. Mice were sacrificed 1 day after Listeria challenge, and the numbers of viable bacteria in the lungs and the spleen were determined as a measure of cellular activation. In separate experiments, bronchoalveolar lavage (BAL) fluid was collected. Only mice exposed to the smallest PSP (0.064 and 0.202 mum) had significantly reduced bacterial numbers in the lung after particles and Listeria were given simultaneously. When particles were given 1 day prior to Listeria challenge also the largest 4.646 mum PSP, but not the medium size 1.053 mum PSP, reduced bacterial numbers. The number of neutrophils in BAL fluid was increased for all PSP-exposed groups after 24 h, and tended to be highest in the group exposed to 4.646 mum PSP. TNF-alpha, IL-1beta and MIP-2 were significantly increased in BAL fluid after exposure to the largest compared with the smallest PSP. In conclusion, activation of the innate immune system by chemical-free particles was size-dependent. Ultrafine and coarse particles appeared to activate cells by different mechanisms, which implies qualitative differences between the health effects of ambient air particulate matter size fractions.

The immune complex transfer enzyme immunoassay: Mechanism of improved sensitivity compared with conventional sandwich enzyme immunoassay.

Immune complex transfer enzyme immunoassay (ICT-EIA) is one of the technologies which enables ultrasensitive measurements of protein biomarkers. The ICT-EIA uses two types of beads and sandwich-shaped immune complexes are transferred from the 1st bead to the 2nd bead in the assay. The purpose of the study is to reveal the reason why the ICT-EIA achieves ultrasensitive measurements by making a detailed comparison between conventional sandwich enzyme immunoassay (Sand-EIA) and ICT-EIA. ICT-EIAs for cytokines were developed and the sensitivities were compared with the sandwich EIAs. ICT-EIAs had about 100 times higher sensitivities because of markedly decreased non-specific signals derived from non-specific binding of detection antibody conjugates onto the polystyrene bead. The results have enabled us to show the importance of reducing non-specific signals in EIAs to obtain higher sensitivities. This methodology should be more valuable if combined with a different label detection system such as digital counting or immuno-PCR, which may enable the detection of single target protein molecules in the near future.

Mechanism of nanoparticle-induced hypersensitivity in pigs: complement or not complement?

A recent study on nanoparticle-induced hypersensitivity reactions in pigs showed robust pulmonary intravascular macrophage clearance of Polybead® carboxylate microspheres in mediating the adverse cardiopulmonary distress, irrespective of the ability of these particles to activate the complement (C) system in vitro. Focusing on this observation, this article highlights the controversies in projecting in vitro C assay data to in vivo conditions and applying data on polystyrene particles to therapeutic nanopharmaceuticals. Based on overwhelming evidence of a role of anaphylatoxins in hypersensitivity reactions, the need to further explore the role of C activation in the reported and other reactions is highlighted. C-activation-related and C-independent pseudoallergies (CARPA and CIPA) can proceed simultaneously, as outlined by the 'double-hit' hypothesis.

Immunogenicity of antigen-conjugated biodegradable polydiacetylene liposomes administered mucosally.

In this work, we report a protocol for synthesizing nanosize ovalbumin-functionalized polydiacetylene (PDA) liposomes (LP-Ova). We show that LP-Ova administered per-orally (p.o.) and subcutaneously (s.c.), without the use of adjuvants, induces high serum IgG1 titers. As reported previously using polystyrene nanoparticles (NPs), p.o.-primed mice developed high titers of IgG2c and intestinal IgA following s.c. boosting immunization with LP-Ova. Mice that received a single s.c. immunization with LP-Ova did not develop serum IgG2c or intestinal IgA antibodies. Additionally, in s.c.-immunized mice serum IgG1 titers decreased significantly by 3 months after immunization. In contrast, in mice primed p.o. and boosted s.c. with LP-Ova, serum IgG1/IgG2c, and intestinal IgA antibody titers remained stable. Administration of LPs exerted no adverse effects on immunized mice as no morbidity or signs of toxicity were observed for the duration of the studies. These results indicate that antigen-conjugated liposomes are immunogenic and confirm a previous report that mucosal priming followed by a s.c. boosting immunization is the most effective strategy for inducing long-lasting mucosal IgA, as well as a polarized Th1/Th2 systemic response. In addition to being biodegradable and easily functionalized by conjugation, liposomes have a hollow core which can also be loaded with cargo, allowing for a targeted delivery of multiple antigens (or drugs) simultaneously. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 557-565, 2017.

Differential macrophage modulation of asymmetric IgG antibody synthesis by soluble or particulate stimuli.

Asymmetric IgG antibodies behave as antigen-blocking due to the presence of a mannose-rich oligosaccharide residue in one of the Fab fragments of the molecule. This feature prevents the interaction of this antibody with the antigenic epitope, then they are unable to activate of the immune effector mechanisms. Taking into account that macrophages modulate the adaptive immune response according to the nature of the stimulating antigen and that IL-6 increases the synthesis of asymmetric IgG, in this work we analysed the differential modulation of the synthesis of asymmetric IgG by culture supernatants from peritoneal macrophages (CSPM) stimulated with either OVA-polystyrene beads or soluble OVA. The levels of IL-10, IL-6 and TNFalpha were determined in CSPM to find elevated levels of IL-6 and TNFalpha only in those cultures stimulated with OVA-coated polystyrene beads. The 112D5 hybridoma was cultured in the presence of CSPM from cells stimulated with OVA-polystyrene beads or soluble OVA. There was an increase in the synthesis of asymmetric IgG only in those cultures of the hybridoma treated with CSPM stimulated with OVA-polystyrene beads this CSPM also had an antiproliferative effect on the hybridoma. A neutralizing anti-IL-6 antibody inhibited the synthesis of asymmetric IgG but could not revert the effect of such CSPM on the viability of the hybridoma. In addition, our results demonstrated that the murine TNFalpha, at a concentration similar to that found in the particle-stimulated CSPM, had an inhibitory effect on cell growth. In summary, IL-6 and TNFalpha are secreted at different concentrations by peritoneal macrophages depending upon the physicochemical nature of the stimulus, thus conditioning the humoral immune response elicited. The IL-6 present in the CSPM can regulate the secretion of asymmetric IgG and, together with TNFalpha can also regulate cell proliferation. Results obtained in this work would contribute to the knowledge of the role of macrophages in the modulation of the quality of the humoral immune response generated upon stimulation with particulate antigens.

Effects of prosthetic materials on the host immune response: evaluation of polymethyl-methacrylate (PMMA), polyethylene (PE), and polystyrene (PS) particles.

The main causes for the long-term prosthetic implants' failure are the body's reaction to the implanted material or mechanical stress on the device resulting in the formation of wear particles. Particulate wear debris attracts macrophages, and depending on the chemical composition of the material and particle size, various levels of inflammatory response may occur. While transient inflammation is common, development of chronic inflammation may have serious consequences, leading to implant failure. Such a process may also cause systemic changes to immune functions and long-term effects on the host immune responses. In this study, we evaluated the effects of polystyrene (PS), polyethylene (PE), and polymethylmethacrylate (PMMA) particles on macrophage function and the generation of T-cell responses. Particles of various diameters were injected intraperitoneally into Balb/c mice, and immune functions were examined at 3, 10, and 21 days after the injection. The intensity of phagocytosis by peritoneal exudate cells (PECs) and the proliferative response of spleen cells from treated mice were evaluated. Enumeration of PECs revealed an increase in the total number of cells. Mice injected with PS or PE particles had a higher percentage of cells containing particles than PMMA-injected mice. Macrophages with PS or PE particles tended to adhere to and/or infiltrate peritoneal fibro-fatty tissues surrounding the spleen and pancreas, while the PMMA-carrying macrophages infiltrated the spleen, resulting in an increase of spleen size and "weight. The spleen cell proliferation assay revealed only mild and transient effects on the mitogen response in both PE and PS particle-injected mice. However, in the PMMA-injected mice we observed a lasting increase of the Con A response and a decrease of the LPS response. In vitro exposure of PECs from untreated mice showed a dose-response pattern in nitric oxide (NO) and TNFalpha production. While exposure to either PMMA or PE induced comparable levels of NO, exposure to PMMA induced a markedly higher production of TNFalpha than exposure to PE. The results indicate that particulate biomaterials may, in addition to the initial activation of phagocytes, significantly affect immune functions and compromise the host response to other antigenic stimuli.

The IgE adjuvant effect of particles: characterisation of the primary cellular response in the draining lymph node.

Diesel exhaust particles, and polystyrene particles (PSP) as a model for the insoluble particle core, have an adjuvant effect on allergen-specific IgE production in mice. We therefore examined the primary immune response in the draining popliteal lymph node (PLN) to the allergen ovalbumin (OVA) injected together with polystyrene particles into the footpad of BALB/cA mice. Similar numbers of particle-containing cells were observed in the draining lymph node on day 1 after injection of PSP alone or OVA + PSP, the numbers increasing continuously until day 21. The total lymph node cell numbers increased three to four times in the OVA + PSP group compared to both OVA and PSP groups, peaking on day 5. The increase in B cell numbers was twice the increase in T cell numbers. On day 5, OVA + PSP increased the expression of most surface markers measured (MHC class II, CD86, CD23, CD69) compared to OVA and PSP. Further, the ex vivo production of IL-4 and IL-10 by PLN cells from OVA + PSP-injected animals was increased. In conclusion, whereas PSP alone did not influence any of the immunologic markers studied, the adjuvant effect of PSP on the IgE antibody response to OVA was associated with an early increased primary cellular response in the draining lymph node.

Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry.

Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-microm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their alpha chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%-19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.

Synthetic antigens. Horse "natural" antibodies against interpolymer of styrene and maleic acid (PSM).

Properties of horse natural anti-PSM antibodies are described. The antibodies were of IgG class. Electrostatic forces were mainly involved in reaction of PSM with horse antibodies. The reaction was inhibited by low molecular compounds resembling structural unit of PSM. Studies of difference spectra and ORD and CD spectra showed no major conformational changes in horse antibodies after reaction with PSM.

Synthetic antigens. Rabbit antibodies against interpolymer of styrene and maleic acid (PSM).

Rabbit antibodies against interpolymer of styrene and maleic acid (PSM) are described. Rabbits immunized with PSM (mol. weight 292,000) produced precipitating and non-precipitating antibodies of IgG class. Interaction of rabbit antibodies with PSM was mainly of electrostatic character. It was found that various low molecular weight compounds resembling structural unit of the polymer inhibited reaction of PSM with antibodies. The reaction was also inhibited by picric acid and epsilon-TNP-aminocaproic acid. It suggested a multifunctional character of antibodies studied. The apparent affinity constant was 2 x 10(5) M-1 for interaction of PSM with the rabbit antibodies. Spectral studies suggested the presence of tyrosine in the combining site of anti-PSM antibodies. Specifically purified antibodies showed no restriction of heterogeneity, in comparison with rabbit IgG containing anti-PSM antibodies.

Synthetic antigens. Antigen-binding cells to interpolymer of styrene and maleic acid (PSM) and sheep red blood cells in mice and rats immunized with PSM.

To study the mechanism of the immune response, a new type of synthetic antigen: interpolymer of styrene and maleic acid (PSM) was introduced in our laboratory. In our previous papers it was proved that PSM is immunogenic and antigenic in various animals PSM had also adjuvant properties.

Interferon induction by SMANCS: a polymer-conjugated derivative of neocarzinostatin.

Poly (styrene-co-maleic acid)-conjugated neocarzinostatin, SMANCS, induced antiviral activities in the circulation of mice. The maximum titer of the activity (240 U/ml) was observed 20 h after administration an 8 mg/kg iv dose of SMANCS. Various experiments showed that this antiviral substance induced by SMANCS was an interferon (IFN). Sixty percent of the IFN response stimulated by SMANCS was impaired in mice pretreated with anti-IFN gamma antiserum. This suggests that the serum IFN induced by the agent contained about 60% of IFN gamma. When Thy 1+ or Lyt 2+ T-cells were depleted from mice by administration of anti-Thy 1.2 or anti-Lyt 2.2 monoclonal antibody (mAb), this IFN response was eliminated. However, when natural killer cells were depleted from mice by treatment with antiasialo GM1 antiserum, no alteration in the IFN response was observed. The SMANCS-stimulated IFN response was also abrogated in mice treated with macrophage blockers (carrageenan and trypan blue), whereas administration of anti-Lyt 1.2 mAb had no effect on the IFN response. These results suggest that macrophage and T-cells w Lyt 1-2+ phenotype may have an important role in the IFN response stimulated by SMANCS.

Fine particles of widely different composition have an adjuvant effect on the production of allergen-specific antibodies.

Diesel exhaust particles (DEP) are reported to increase the specific IgE response to allergens, and results from our laboratory suggest that the particle core of DEP contribute to this adjuvant activity. The purpose of the present study was to explore further the adjuvant effect of particles per se, that is particles by themselves. NIH/Ola mice were given two intraperitoneal injections with ovalbumin (OVA; 10 microg) alone or OVA in combination with PSP, polytetrafluoroethylene (teflon), titanium dioxide (TiO(2)) or amorphous silica particles (2.8x10(10)-2.8x10(12)). Blood samples were drawn 7 days after the last injection, and serum levels of allergen-specific and total IgE and IgG2a were measured. All types of particles gave increased levels of allergen-specific IgE and IgG2a. Similar results were obtained after intranasal or intratracheal instillation with OVA plus PSP or silica. Our results indicate that fine particles of widely different composition may have an adjuvant effect on the production of allergen-specific antibodies.

Beta-trace protein test: new guidelines for the reliable diagnosis of cerebrospinal fluid fistula.

OBJECTIVE: Cerebrospinal fluid (CSF) fistulas need to be reliably diagnosed for the optimal management. Recently, in preference to beta2-transferrin, another CSF protein, beta-trace protein (betaTP), is similarly used with a new method for CSF diagnosis. This study evaluates the sensitive interpretation and limits of this new betaTP test for use in routine CSF fistula diagnosis. METHODS: Nephelometric detection of betaTP has been made in nasal secretion, serum, and CSF samples from healthy individuals as well as patients with reduced glomerular filtration rate and with bacterial meningitis. Additionally, 53 patients with suspected CSF rhinorrhea are also analyzed. RESULTS: The betaTP test can also be used to reliably diagnose CSF rhinorrhea even slightly better than the beta2-transferrin test. It should not be used for patients with renal insufficiency and bacterial meningitis as they substantially increase serum and decrease CSF betaTP values, respectively. CONCLUSION: Quantitative measurement of betaTP is a noninvasive, highly sensitive, quick, and inexpensive method that can be used for the detection of CSF rhinorrhea in nasal secretions. However, in cases where there is doubt about the interpretation, the results should be proved with beta2-transferrin test or sodium-fluorescein test.