Richard J. Kowalski

Assessing relative risks of infection and rejection : A meta-analysis using an immune function assay

Background. Long-term use of immunosuppressants is associated with significant morbidity and mortality in transplant recipients. A simple whole blood assay that has U.S. Food and Drug Administration clearance directly assesses the net state of immune function of allograft recipients for better individualization of therapy. A meta-analysis of 504 solid organ transplant recipients (heart, kidney, kidney-pancreas, liver and small bowel) from 10 U.S. centers was performed using the Cylex ImmuKnow assay. Methods. Blood samples were taken from recipients at various times posttransplant and compared with clinical course (stable, rejection, infection). In this analysis, 39 biopsy-proven cellular rejections and 66 diagnosed infections occurred. Odds ratios of infection or rejection were calculated based on measured immune response values. Results. A recipient with an immune response value of 25 ng/ml adenosine triphosphate (ATP) was 12 times (95% confidence of 4 to 36) more likely to develop an infection than a recipient with a stronger immune response. Similarly, a recipient with an immune response of 700 ng/ml ATP was 30 times (95% confidence of 8 to 112) more likely to develop a cellular rejection than a recipient with a lower immune response value. Of note is the intersection of odds ratio curves for infection and rejection in the moderate immune response zone (280 ng/ml ATP). This intersection of risk curves provides an immunological target of immune function for solid organ recipients. Conclusion. 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.

Immune cell function testing: An adjunct to therapeutic drug monitoring in transplant patient management

Abstract:  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.

Establishing pediatric immune response zones using the Cylex® ImmuKnow assay

Abstract:  For all transplant patients, the transplant physician must balance the risk of rejection caused by under‐immunosuppression against the risk of drug toxicity, secondary infections and post‐transplant lymphoproliferative disorder with over‐immunosuppression. A Food and Drug Administration (FDA)‐approved in vitro assay, the Cylex® ImmuKnowTM assay, provides a global assessment of cellular immune function to help monitor the immune status of immunosuppressed patients. This assay uses the plant lectin phytohemagglutinin to stimulate lymphocytes; an ATP assay is then used to measure the degree of activation of CD4+ T cells. However, the normal values for this assay were developed with healthy adult patients. In this study, we determined the normal ranges for the ImmuKnowTM assay in healthy children and compared those values to levels obtained in healthy adults and in stable pediatric renal transplant patients. We found that healthy children 12 yr of age and older showed immune function levels indistinguishable from adults, while healthy children under 12 had significantly lower immune function levels than adults. For adults, the ImmuKnowTM assay zones (in ng/mL ATP) of strong, moderate and low immune function correspond to >525, 225 to 525, and <225. In children under 12, we found the corresponding zones to be >395, 175–395 and <175 ng/mL. The median value for normal adults is 415, whereas it is only 295 for children <12 yr of age and this value decreases to 165 in stable renal transplant patients <12 yr of age (compared with 258 for stable adult renal transplant patients). Thus, this study provides critical information necessary to utilize the ImmuKnowTM assay with pediatric patients. In adults, the degree of immune function as assessed by the ImmuKnowTM assay helps to predict patients at risk for infection or rejection. If further studies in pediatric patients document the same and is true for children, then the ImmuKnowTM assay will provide a useful adjunct tool to prevent over‐ or under‐immunosuppression as newly developed drugs are utilized or drug treatment is altered because of drug side effects, toxicity, concurrent illnesses or rejection.

Pancreas transplantation under alemtuzumab (Campath-1H) and tacrolimus: Correlation between low T-cell responses and infection

Background. Alemtuzumab induction and tacrolimus-based immunosuppression has been effective in pancreas transplantation. Despite the encouraging results of this minimalistic approach to immunosuppression, infection still remains a significant cause of morbidity. The Cylex ImmunoKnow assay was used in this study to compare pancreas recipient clinical states (stable, rejection, infection) with T cell responses. Methods. Blood samples were taken from pancreas recipients pretransplant and at approximately three-month intervals posttransplant for analysis of T cell responses. When possible, T cell responses were also quantified during changes in clinical status (infection or rejection). Results. A range between 100–300 ng/ml adenosine triphosphate (ATP) was found in stable patients (mean 194±123, n=51) with good graft function and no infection or rejection. A low T cell response was highly correlated with infectious states. The fourteen patients with infections/posttransplant lymphoproliferative disease had a mean ATP of 48 ng/ml. Risk hazard analysis showed that patients with ATP levels <100 ng/ml were four to seven times more susceptible to infection compared to stable patients. Four patients with rejection showed a T cell response of 550 ng/ml ATP, which was statistically significant compared to stable patients, although the sampling numbers (9) were too small to be conclusive. Conclusion. The Cylex ImmunoKnow assay is a valuable tool to more precisely modulate immunosuppression in pancreas transplant patients. In particular, the assay is extremely useful in detecting overly immunosuppressed patients vulnerable to infections.

Immunodiagnostics: Evaluation of Functional T-Cell Immunocompetence in Whole Blood Independent of Circulating Cell Numbers

The need for a systematic approach for immune function monitoring has becoming increasingly apparent in the past decade due to the rapid expansion of the development and use of immunomodulatory drug therapies and vaccines. While there has been a great deal of progress in the development of methodologies for evaluating and enumerating T-lymphocyte responses to infection and cancer, the translation of these assays into the clinical setting has remained seemingly elusive. This is likely due to inherent difficulties in the standardization and validation of cell-based assays. Here, we describe a novel assay that measures ATP production in CD4+ T-lymphocytes in response to stimulation. Results from the test, unlike absolute cell counts, assess the functional response of lymphocytes. Clinical utility of the assay has been demonstrated in managing immunosuppression in solid organ transplant recipients such that adverse events such as infection and rejection can be avoided. The need for a global immune response test in the clinical setting of transplantation and the value of such a test in post-transplant management is discussed. Furthermore, additional applications of this assay for monitoring diseases that impact immune function including autoimmunity and infection are considered.

Measuring Immune Function in Non-Hodgkins Lymphoma Patients Using the Cylex Immune Cell Function Assay

Patients Using the Cylex Immune Cell Function Assay Karel Dicke, Diane Post, Juliana Woodcock, Richard Kowalski. Arlington Cancer Center, Arlington, TX; Cylex Incorporated, Columbia, MD. Cylex has developed a non-invasive assay to quantify global immune function. The Cylex Immune Cell Function Assay (ImmuKnow), cleared by the FDA for assessing cell-mediated immunity in immunosuppressed populations, measures T cell activation in whole blood following in vitro exposure to PHA for 15–18 hours. The clinically defined zones of immune response, as measured by quantifying ATP after stimulation, consist of strong (.524 ng/mL), moderate (226–524 ng/mL) and low (,226 ng/mL). In this pilot study, 7 patients with non-Hodgkins lymphoma were tested. The average immune response for these patients was 310 ng/mL, which is significantly lower (p 1⁄4 0.0019) than apparently healthy volunteers (615 ng/mL ATP) tested in conjunction with the cancer patients. Individual responses in the cancer patients tested ranged from 129 ng/mL-598 ng/mL ATP (Table 1). Two of the patients, JB and EB, with very low immune response (129 and 163 ng/mL ATP respectively) had active recurrence of their lymphoma at the time blood was drawn for ImmuKnow testing. Patient EB had radiation treatment two weeks prior to time of test. Conversely, the patient, MW, with a high immune response (598 ng/mL ATP) had no evidence of lymphoma at the time the sample was tested. These preliminary results suggest that the Cylex ImmuKnow assay is a useful tool for monitoring a cancer patient’s response to therapy and long-term prognosis for cancer remission or recurrence.