These data show that the Cylex ImmuKnow assay has a high negative predictive value and provides a target immunological response zone for minimizing risk and managing patients to stability.
Each year, 55 000 organ transplants are performed worldwide. Cumulatively, the number of living organ recipients is now estimated to be over 300 000. Most of these transplant recipients will remain on immunosuppressive drugs for the remainder of their lives to prevent rejection episodes. Controlled doses of these drugs are required to prevent over-medication, which may leave the patient susceptible to opportunistic infection and drug toxicity effects, or under-dosing, which may lead to shortened graft survival because of rejection episodes. This paper describes the result of a multicenter study conducted at the Universities of Pittsburgh, Alabama and Maryland to evaluate an in vitro assay (CylexTM Immune Cell Function Assay) for the measurement of global immune response in transplant patients receiving immunosuppressive therapy. The assay uses a whole blood sample to maintain the presence of the drug during incubation. Following overnight incubation of blood with phytohemagglutinin (PHA), CD4 cells are selected using paramagnetic particles coated with a monoclonal antibody to the CD4 epitope. The CD4-positive cells are targeted as major immunosuppressive drugs are designed to specifically inhibit T-cell activation which has been implicated in rejection. The data generated at these three sites were submitted in support of an Food and Drug Association (FDA) application for the use of this assay in the detection of cell-mediated immunity in an immunosuppressed population. The assay was cleared by the FDA on April 2, 2002. This cross-sectional study was designed to establish ranges for reactivity of this bioassay in the assessment of functional immunity for an individual solid organ recipient at any point in time.
The proliferative response is most frequently determined by estimating the amount of Cell proliferation assays have been shown to be useful for monitoring T-cell immune status. Similarly, T-cell activation, as measured by determining the increase in intracellular ATP, has been correlated with proliferation. After an initial period of consumption (10), ATP levels increase, and a linear relationship between cell concentration and ATP level which is proportional to light intensity develops. This measured luminescence has been favorably correlated with cell number (1,5,6,9) and the degree of activation of peripheral blood mononuclear cells (3, 4) in cell proliferation studies.Traditionally, researchers have purified peripheral blood mononuclear cells prior to stimulation, a method requiring careful handling and multiple centrifugation steps (2). More recently, whole blood has been successfully used for these types of studies (7,8). Whole blood is particularly useful when a large number of blood samples must be processed on a given day, when blood volume is limited, or when facilities and expertise are limited.Proliferation testing is not widely used in clinical settings because of the relatively long period of time needed to achieve results (3 to 10 days), the lack of assay standardization, and the requirement for the use of isotopes. This paper describes an ATP assay for measuring T-cell activation which can utilize whole blood, achieve results in 24 h, does not involve the use of isotopes, and has the additional advantage of identifying the specific T-cell subset involved. The Luminetics assay of T-cell activation, supplied in a kit format which includes diluents, wash buffers, monoclonal-antibody-coated beads, standards, and controls, is suitable for use in a clinical laboratory.To compare the ATP assay of cell activation with the cell proliferation assay ([ 3 H]thymidine incorporation), T-lymphocyte responses to mitogens and antigens in microcultures of whole blood from healthy adult humans, obtained before and after booster vaccination with tetanus toxoid (TT) and diphtheria toxoid, were measured.Ten healthy adults were bled two times at a 6-week interval. Immediately after the first blood collection (P1), six individuals were given a booster vaccination of TT and diphtheria toxoid.Four subjects were not administered the booster vaccine. Six weeks after the first blood collection, all subjects were bled a second time (P2). Both test methodologies were initiated within 4 h after blood collection. Whole blood was collected in Vacutainer tubes containing 45 USP units of sodium heparin (Becton-Dickinson, Rutherford, N.J.) and held at room temperature until processed, approximately 2 to 3 h. The blood was diluted 1:4 with RPMI 1640 medium (Gibco BRL). Phytohemagglutinin L (PHA-L; Sigma Chemical Co.), concanavalin A (ConA; Sigma Chemical Co.), and purified TT (Connaught Laboratories, Swiftwater, Pa.) were diluted in RPMI 1640 and used in both test systems.In the ATP assay method, 100 l of 1:4-diluted blood was added ...
Profound T-cell depletion with the monoclonal antibody alemtuzumab facilitates reduced maintenance immunosuppression in abdominal and lung transplantation. While the phenotype of the post-depletional T cells has been characterized, little is known about their function. In the present study, global and CMVspecific T-cell function was assessed longitudinally in 23 lung transplant (LTx) recipients using T-cell assays (ImmuKnow ® and T Cell Memory TM , Cylex, Columbia, MD) during the first year posttransplant after induction therapy. Recovery of mitogen responses were seen at 2 weeks posttransplantation (65%PHA; 58% Con A), despite the low number of circulating T cells (<2%). These responses declined at 4-5 months (24%PHA; 54% Con A) and were partially reconstituted by 9 months (46% PHA; 73% Con A). CMV-specific responses recovered in 80% of R+ patients as early as 2 weeks posttransplant (n = 5) and 72% of patients had a memory response by 3 months (n = 11). In contrast, only 2 of 5 patients who did not exhibit memory responses pre-transplant (R-) developed transient CMV-specific T-cell responses. Our results show that profound depletion of T cells induced by alemtuzumab spares the functional subset of CMV-specific memory T cells. Conversely, CMV R-patients predepletion may require a prolonged period of prophylaxis.
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