Acute cardiac rejection may be difficult to diagnose in orthotopic cardiac transplant patients. The endomyocardial biopsy is the current gold standard for detection of acute rejection. This study examined the feasibility of morphologic evaluation of peripheral blood lymphocytes to provide a sensitive indicator of acute cardiac rejection.
Material and Methods
Peripheral blood lymphocytes of 49 orthotopic cardiac transplant patients were repeatedly examined over three years. Immunosuppressive therapy for these patients included preoperative antithymocyte globulin, followed by an intraoperative dose of methylpred-nisone and antithymocyte globulin. The postoperative regimen consisted of individualized doses of horse or rabbit antithymocyte globulin, cyclosporine A, prednisone and azathioprine. Rejection episodes were treated with methylprednisone; refractory acute rejection was rarely treated with the mouse monoclonal antibody OKT3.
Cytologic studies of peripheral lymphocytes were performed at the end of the first postoperative week, then biweekly until discharge. After discharge, cytologic studies were performed at 3, 4.5, 6, 9, and 12 months, then every 3 months thereafter. Endomyocardial biopsy study results were correlated with simultaneous cytologic studies. Peripheral blood lymphocyte studies were also obtained whenever a patient presented with clinical symptoms suspicious for acute cardiac rejection. medicated eye drops
A 10-ml aliquot of heparinized whole blood was purified on a Ficoll Hypaque gradient. A cytospin preparation of concentrated, purified lymphocytes was then stained with a Wright-Giemsa stain. Lymphocytes were measured with a calibrated microscope and defined as “activated” if they measured greater than 10|x in diameter and possessed a prominent nucleolus. Azurophilic or granular cytoplasm was noted when present. If a patient had more than 30 activated lymphocytes/cu mm, the lymphocytes were classified as “immune activated.” The lymphocytes from patients were then stained with the monoclonal antibodies Leu 3a (T-helper/inducer cells), Leu 2a (cytotoxic/suppressor cells), Leu 4 (mitogenic T cells), Leu 5b (E rosette receptor associated antigen), Leu 12 (B cells), or Leu 2a+15_ (cytotoxic cells) and analyzed via flow cytometry (Becton-Dickinson Monoclonal Center, Inc). Absolute numbers of T cells, B cells, and helper/suppressor ratio (Leu 3a/2a) were correlated retrospectively with endomyocardial biopsy results and morphologic lymphocyte studies. A Students t test was performed on absolute numbers of the above-described lymphocytes to evaluate significant differences during acute rejection.-2 Sensitivity, specificity, and positive and negative predictive values were determined as described by Weinstein and Fineberg.
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