Performance of a novel BD stem cell enumeration kit on two flow cytometry systems.

INTRODUCTION: Hematopoietic stem cell transplantation, which requires accurate enumeration of stem cells, is routinely used in clinical settings. Flow cytometry provides a qualitative and quantitative assessment of CD34⁺ cells. Precision, linearity, and stability of the novel BD™ Stem Cell Enumeration (SCE) Kit were evaluated on two flow cytometry platforms using a modified ISHAGE gating strategy and including a viability dye for data acquisition and analysis. METHODS: Precision and linearity were evaluated on BD FACSCanto™ II and BD FACSCalibur™ systems. Stability was evaluated on the BD FACSCanto II system. Precision was tested using both high and low controls. Linearity was evaluated using dilutions from CD34⁺ cell pools, while stability was evaluated using fresh leukapheresis specimens. RESULTS: Both systems showed precision with limited variability in absolute counts and percentages of viable CD34⁺ cells. The linearity range of viable CD34⁺ cells in both systems was established at 0-1000 cells/µL, showing a linear relationship (R² = 0.99). Stability of CD34⁺ cells in mobilized leukapheresis samples was confirmed up to 24 h after collection and up to 60 min after the end of stain/lyse procedures. CONCLUSION: The BD SCE Kit on both flow cytometry systems shows consistent and reproducible results.

Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.

The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.

Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics.

Tears play an essential role in maintaining corneal and conjunctival integrity by providing a tightly regulated, optimal extracellular environment critical to its numerous functions, which include anti-microbial defense, wound healing and inflammatory responses such as allergies. Elevated levels of inflammatory cytokines have been reported in tears from various ocular disease states. Characterization of tear cytokines has been limited by the small volume (microliter amounts) attainable. This limitation was addressed with the newly developed Becton Dickinson Cytometric Bead Array (CBA), which combines the principles of the "sandwich" immunoassay with the capability of flow cytometry for simultaneous measurement of the characteristics of multiple particles. This technique allows determination of six human cytokine (IFNgamma, TNFalpha, IL-2, IL-4, IL-5, IL-10) concentrations simultaneously in a single tear sample. Tears were collected from the inferior fornix of non-allergic (n=7) and allergic (n=9) donors. Each tear sample or cytokine standard was incubated with a mixture of capture Ab-bead reagent and detector Ab-phycoerythrin (PE) reagent, and analyzed using flow cytometry. All six cytokines were detectable in both non-allergic and allergic tears. Tears from allergic donors contained significantly less IL-10 (p=0.035), and had significant increases in the ratios of TNFalpha/IFNgamma, IL-5/IFNgamma and IL-5/IL-10 (p=0.0008, 0.0124 and 0.011, respectively). The small volume required (5-10 microl/test) by the Cytometric Bead Array allows measurement of all six cytokines from a single collection of tears. This decreases collection time, minimizing the confounding effect of stimulation on cytokine concentration in tears, as well as allowing calculation of cytokine ratios.

Vaccines against Pseudomonas aeruginosa infection: 2. Clinico-immunological investigations.

A new multicomponent Pseudomonas vaccine 'pyoimmunogen' has been developed. It was tested for safety and immunogenicity by immunization of volunteer donors and burned patients. Immune plasma obtained from volunteers was used for treatment of severe forms of P. aeruginosa infections. Pyoimmunogen was shown to be of low toxicity for apparently healthy humans and to induce specific antibody formation. In 16 out of 20 burned patients it had a beneficial effect on the course of the disease and the period of hospitalization was shortened. As a result of complex treatment involving the use of anti-P. aeruginosa plasma 51 out of 60 patients recovered.

[Immunoenzyme analysis method for detecting anti-Pseudomonas aeruginosa antibodies in persons inoculated with pyoimmunogen]. / Metod immunofermentnogo analiza dlia vyiavleniia antisinegnoinykh antitel u lits, privitykh pioimmunogenom.

The possibility of using the indirect ELISA techniques for evaluating the level of the post-vaccinal production of humoral antibodies in donors immunized with Pyoimmunogen. P. aeruginosa vaccine, has been studied. The specificity and high resolution of this test system, based on the immobilization of the antigens of the vaccinal preparation on a solid-phase carrier, have been demonstrated. A rational method for the evaluation of specific antibody titers with due regard to the spectrophotometric data indicating the results of the reaction and the degree of the dilution of the serum under test has been proposed.

The effect of transfer factor on spontaneous shedding of sheep red blood cell binding receptors of T lymphocytes in vitro.

Many effects of transfer factor can be used for testing of its activity in vitro. Its effect on rosette formation has been utilized in two methods: the enhancement of rosetting of trypsin-treated T lymphocytes and the increase of 'active' rosettes depressed under some immunopathological conditions. 'Active' rosetting lymphocytes of healthy blood donors if kept at 37 degrees C for 4 hr shed partly their sheep red blood cell-binding receptors into the culture medium supplemented with 25% fetal calf serum. The adding of the negative skin test-converting fraction of dialysable leucocyte extracts inhibits the decrease of the number of rosettes. Possible explanations for the observed phenomenon are: transfer factor increases the rate of receptor synthesis, it causes uncovering or redistribution of the receptors, or it stabilizes otherwise shed membrane structures.

Interaction of targeted liposomes with human peripheral mononuclear blood cells and lymphocytes of chronic lymphoid leukaemic patients.

Association of small unilamellar vesicles (SUV) can be effectively targeted to normal human peripheral mononuclear blood cells (PMBC). - Lymphocytes of chronic lymphoid leukaemic (CLL) patients show a low rate of liposome association, which cannot be improved by targeting with anti-lymphocyte globulin (ALG). Presence of serum evoked an enhanced association of liposomes to PMBC.

Synthesis and studies of transferrin.

Combination of the usual techniques yielded 70-80 p.c. pure apo- and iron-transferrin preparations, whose analysis revealed the presence of antigenically identical transferrin fractions differing from one another in respect of electrophoretic motility and sedimentation contrast. No similar modifications of transferrin have previously been reported.

Characterization of enhancing factor (EF) prepared from spleen of mice immunized with tetanus toxoid.

In cultures of spleen cells from tetanus toxoid-primed mice a soluble, nonspecific material appeared, which enhanced antibody response in vivo. The active material was purified by gel filtration on Sephadex-G-150 and by affinity chromatography on Con-A lectin. According to the immunological and physiochemical investigation the active material does not contain carbohydrate and its molecular weight is in the 100 000 dalton range.

Hydrophobic interaction of extracellular protein A from Staphylococcus aureus with octyl-sepharose CL-4B.

Extracellular Protein A from Staphylococcus aureus was shown to bind to alkyl agarose adsorbents of different carbon chain length. Octyl-Sepharose CL-4B was chosen to purify the active material, as the strong interaction with this adsorbent allowed the separation of Protein A from the bulk of the contaminants. Protein A retained its ability to bind to IgG in the presence of up to 3.5 M ethylene glycol as demonstrated by hemagglutination. A theory on the possible involvement of hydrophobic interaction in the binding reaction of Protein A to IgG is advanced.

The influence of enzymatic cleavage and chemical modification of human and rabbit IgG on their reactivity with staphylococcal protein A.

Proteolytic cleavage fragments from rabbit IgG have been isolated and characterized in an attempt to locate the sites involved in the reactivity with Staphylococcal protein A. The plasmin cleavage product Facb together with the pepsin cleavage products F(ab')2 and pFc' failed to react in contrast to the papain Fc fragment. These data, together with data from unfractionated plasmin digests, in which the Facb fragment remains associated with the plasmin pFc' fragment, indicate that inter-domain interactions are important in the maintenance of this activity. beta2-microglobulin was also shown to be unreactive with protein A. Chemical modification studies employing flurescamine, tetranitromethane and potassium cyanate indicate that lysine and tyrosine residues are not involved in the reactivity of human and rabbit IgG with protein A.

Separation of beta2-microglobulin from transfer factor in dialyzable leukocyte extracts.

Dialyzable leukocyte extracts prepared according to the original method of LAWRENCE contain 4 X 10(4) molecules of beta2-microglobulin per lymphocyte equivalent. The negative skin test converting biological activity (transfer factor) is separable by means of Sephadex G-25 gel chromatography from the beta2-microglobulin component of the extracts. This finding does not support the hypothesis of SHIFRINE and SCIBIENSKI on beta2-microglobulin being the nonspecific anchor of specific transfer factor to nonsensitized lymphocytes.

Application of pepsin-digested horse antibodies for quantitative immunoelectrophoresis.

The majority of the precipitating antibodies in hyperimmune horse serum belong to the beta globulins, as demonstrated by reversed immunoelectrophoresis. As these antibodies migrate in agarose gel during electrophoresis in conventional pH = 8.6-8.9 buffers, horse antiserum cannot be used satisfactorily for quantitative immunoelectrophoresis. On pepsin digestion of horse antiserum F(ab)' 2 fragments are generated which migrate with the gamma globulins. This cleaved product works well in quantitative immunoelectrophoretic techniques.

Biochemical, immunochemical and immunobiological properties of a chloramphenicol-resistant Salmonella typhi strain isolated in Mexico.

The authors have compared the biochemical, immunochemical and immunobiological properties of a chloramphenicol-resistant S. typhi strain isolated during the typhoid fever epidemic in Mexico of 1972 and those of a chloramphenicol-sensitive strain (S. typhi Ty2). They have found no difference in the chemical composition of the two strains. The immunobiological investigations have shown, that the chloramphenicol-resistant strain contains less Vi antigen, it is less virulent on mice, and its active and passive mouse-protective ability is lower than that of the chloramphenicol-sensitive S. typhi Ty2 strain. These findings supported by the result of the electrophoretic analysis suggest that the chloramphenicol-resistant strain is a VW strain, while the chloramphenicol-sensitive strain is a full V strain; the quantitative difference found between the immunogenicity of the two strains to the advantage of the chloramphenicol-sensitive strain may probably be explained by this fact. On the basis of their immunological investigations the authors are of the opinion, that vaccines prepared from chloramphenicol-sensitive S. typhi strains (provided these are full V strains, as e.g. the S. typhi Ty2 strain) will protect most likely also man against infection due to chloramphenicol-resistant S. typhi strains.