ImmuKnow Levels Research Articles

Pre-transplant immune cell function assay as a predictor of early cardiac allograft rejection.

ImmuKnow, an immune cell function assay that quantifies overall immune system activity can assist in post-transplant immunosuppression adjustment. However, the utility of pre-transplant ImmuKnow results representing a patient's baseline immune system activity is unknown. This study sought to assess if pre-transplant ImmuKnow results are predictive of rejection at the time of first biopsy in our cardiac transplant population. This is a single center, retrospective observational study of consecutive patients from January 1, 2018 to October 1, 2020 who underwent orthotopic cardiac transplantation at NYU Langone Health. Patients were excluded if a pre-transplant ImmuKnow assay was not performed. ImmuKnow results were categorized according to clinical interpretation ranges (low, moderate, and high activity), and patients were divided into two groups: a low activity group versus a combined moderate-high activity group. Pre-transplant clinical characteristics, induction immunosuppression use, early postoperative tacrolimus levels, and first endomyocardial biopsy results were collected for all patients. Rates of clinically significant early rejection (defined as rejection≥1R/1B) were compared between pre-transplant ImmuKnow groups. Of 110 patients who underwent cardiac transplant, 81 had pre-transplant ImmuKnow results. The low ImmuKnow activity group was comprised of 15 patients, and 66 patients were in the combined moderate-high group. Baseline characteristics were similar between groups. Early rejection occurred in 0 (0%) patients with low pre-transplant ImmuKnow levels. Among the moderate- high pre-transplant ImmuKnow group, 16 (24.2%) patients experienced early rejection (P=.033). The mean ImmuKnow level in the non-rejection group was the 364.9ng/ml of ATP compared to 499.3ng/ml of ATP for those with rejection (P=.020). Patients with low pre-transplant ImmuKnow levels had lower risk of early rejection when compared with patients with moderate or high levels. Our study suggests a possible utility in performing pre-transplant ImmuKnow to identify patients at-risk for early rejection who may benefit from intensified upfront immunosuppression as well as to recognize those where slower calcineurin inhibitor initiation may be appropriate.

Pre-Transplant Immune Cell Function Assay as a Predictor of Early Cardiac Allograft Rejection

Purpose ImmuKnow, an immune cell function assay that quantifies overall immune system activity can assist in post-transplant immunosuppression adjustment. The utility of pretransplant ImmuKnow results representing a patient's baseline immune system activity is unknown. We sought to assess if pretransplant ImmuKnow results are predictive of rejection at the time of first biopsy in a cardiac transplant population.. Methods 81 cardiac transplant patients with pre-transplant ImmuKnow results (out of 110 total transplant patients) were identified. ImmuKnow results were analyzed according to the clinical interpretation ranges (low, medium, high activity). Pre-transplant clinical characteristics, induction immunosuppression use, early postoperative tacrolimus levels (Day 7 post transplant), and first endomyocardial biopsy results were collected for all patients. Logistic regression was used to estimate the association of the pre-transplant ImmunKnow group with clinically significant early rejection (≥ 1R/2), and to determine if the association was modified by the use of induction therapy and early tacrolimus levels. Results Significant early rejection was found in 15 patients (18.5%). Early rejection occurred in 0/15 patients with low pretransplant ImmuKnow levels vs. 15/64 patients with moderate or high pre-transplant ImmunoKnow levels (p=0.04). Induction immunosuppression was used for 26 patients (32.1%). The most common reason for induction was dual organ transplantation. Of the 55 patients without induction immunosuppression, 13 had rejection (23.6%). The mean ImmuKnow level in the non-rejection group was 391 ng/ml of ATP compared to 516 ng/ml of ATP for those with rejection (p=0.06). Lower pretransplant ImmuKnow levels among non-rejection patients was not diminished when controlling for tacrolimus level. Conclusion Patients with low pre-transplant ImmunoKnow levels had a lower risk of early rejection when compared with patients with medium or high levels. After adjusting for tacrolimus level, this relationship was maintained. Our study suggests a possible utility in performing pretransplant ImmunoKnow to identify patients at-risk for early rejection who may benefit from intensified upfront immunosuppression.

CD4 T-cell function assay using Cylex ImmuKnow and lymphocyte subset recovery following allogeneic hematopoietic stem cell transplantation

We analyzed CD4 T-lymphocytes ATP levels along with lymphocyte subsets in 160 samples from 111 post-allogeneic hematopoietic stem cell transplantation (alloHSCT) patients. In patients with stable status, ImmuKnow levels changed over time and the 6-month post-alloHSCT levels were significantly higher than those tested within 6months post-alloHSCT (P<0.001). Although, ImmuKnow levels for acute graft-versus-host disease (GVHD) or infection episodes were not significantly different compared to those for stable alloHSCT, the levels were correlated with specific lymphocyte subpopulations at different times; the results within 6months post-alloHSCT showed low positive correlation with natural killer cell count (r=0.328) (P<0.05) and the values tested later than 6months post-alloHSCT were positively correlated with CD4 T cell count (r=0.425) (P<0.05). Two patients who developed acute GVHD and two who experienced an infection episode showed increased ImmuKnow levels in sequential tests. The combined test of ImmuKnow levels and lymphocyte subsets may be helpful for immune monitoring following alloHSCT.

Evaluation of the immune function assay in pediatric living donor liver transplantation.

The immune function (ImmuKnow) assay is a measure of cell-mediated immunity based on the peripheral CD4+ T cell ATP activity. The efficacy of ImmuKnow in pediatric LDLT is not well documented. The aim of this study was to assess the correlations between the ImmuKnow and the clinical status in pediatric LDLT recipients. A total of 716 blood samples were obtained from 60 pediatric LDLT recipients (one month to 16 yr of age). The recipient's status was classified as follows: stable, infection, or rejection. The ImmuKnow values in the pediatric LDLT recipients with a clinically stable status had a lower immune response (IQR 85-297 ATP ng/mL) than that previously reported in adults. Meanwhile, the ImmuKnow values of the stable patients were not correlated with age. Furthermore, a significant difference was found in the ImmuKnow values between the bacterial or fungal infection and stable groups, but not between the CMV or EBV infection and stable groups. The ImmuKnow levels in the pediatric LDLT were lower than those observed in the adult LDLT. The proposed reference value is between 85 and 297 ATP ng/mL in pediatric LDLT recipients. We conclude that the ImmuKnow assay could be helpful for monitoring pediatric LDLT recipients with bacterial or fungal infections.

Usefulness of Monitoring Cell-Mediated Immunity for Predicting Post–Kidney Transplantation Viral Infection

BackgroundMonitoring cell-mediated immunity (CMI) can be used to estimate the risk of viral infections in kidney transplant recipients. The Immuknow (IMK) assay measures CD4+ T-cell adenosine triphosphate activity, assesses patient CMI status, and assists clinicians in determining the risk of viral infection. MethodsWe retrospectively analyzed 224 IMK values in 39 kidney transplant recipients at our institution from April 2012 to January 2013. We analyzed the relationship between IMK value and viral infection during the early and late post-transplantation periods. Multiple regression analyses were performed, to determine which factors impacted the results of the IMK assay. ResultsEight patients developed viral infections, including BK virus, cytomegalovirus, herpes simplex, and shingles. Five infections occurred in the early post-transplantation period (<50 d) and 3 in the late period (>120 d). The IMK levels in patients who developed an infection in the early period were within normal limits; however, those in the late period were significantly lower than 200 ng/mL (421.0 ± 062.6 for early vs 153.7 ± 72.7 for late; P = .02).Our multiple regression analyses indicated that peripheral white blood cell and neutrophil counts affected IMK values (P = .03 and P = .02, respectively). ConclusionsThe IMK assay is a useful test for identifying patients at risk for post-transplantation viral infections in the late transplant period.

Lack of clinical association and effect of peripheral WBC counts on immune cell function test in kidney transplant recipients with T-cell depleting induction and steroid-sparing maintenance therapy

The Cylex ImmuKnow assay measures the amount of stimulated ATP production by CD4+ T-cells, and has been used clinically, trying to predict rejection and infection episodes. However, predictive values of this assay after induction therapy with steroid-sparing maintenance protocols are unclear. In this single-center cohort study, we analyzed renal transplant recipients who received T-cell depleting+/−anti-IL2 receptor antibodies and tacrolimus/mycophenolate maintenance without steroids. A total of 4224 ImmuKnow levels in 306 patients were available for analysis. ImmuKnow levels (Mean±SE) changed over time after induction therapy with a paradoxical initial increase: 419±23, 461±32, 519±14, 411±10, 344±6, and 405±3 for pre-transplant, 0–1wk, 1wk–1mo, 1–3mos, 3mos–1yr, and thereafter. This change was parallel to the evolution of peripheral WBC counts and ImmuKnow levels had weak but significant correlation with WBC counts (R2=0.264, P<0.0001). The levels for biopsy-proven rejection (389±56) and borderline/clinical rejection (254±41) were not significantly higher than the levels of quiescent patients. The levels for opportunistic infection (349±48) and other infections (345±27) were not significantly lower than the levels of quiescent patients. The longitudinal changes in ImmuKnow levels were not predictive of rejection or infection. In conclusion, ImmuKnow levels can vary after T-cell depleting induction therapies at various time points, even without significant clinical events. Since ImmuKnow levels seem to be affected by WBC counts, ImmuKnow results need to be interpreted with caution. The effects of leukocytosis or leukopenia caused by immunosuppressive medication on the ImmuKnow assay need further investigation.

Cellular immune function monitoring after allogeneic haematopoietic cell transplantation: evaluation of a new assay

Managing the patient's immune system after haematopoietic cell transplantation (HCT) is a challenge, mainly in the unstable period immediately after the transplant. Currently there is no standardized non-invasive diagnostic tool for the evaluation of immunological complications such as graft-versus-host disease (GVHD) and for managing the cellular immune function of the transplant recipient. The ImmuKnow assay for cellular immune function monitoring has been incorporated successfully into the clinical follow-up routine of solid organ transplant recipients. This study aims to explore the relevance and potential contribution of immune monitoring using the assay in the setting of HCT. We found that ImmuKnow-level measurement can distinguish between states of immune function quiescence and between events of acute GVHD. ImmuKnow levels were significantly higher in patients going through GVHD than the levels measured for the same patients during immunological stability. Moreover, we demonstrate a patient case where longitudinal monitoring using the ImmuKnow assay provided a trustworthy depiction of the patient's cellular immune function post-HCT. In conclusion, we provide evidence for the potential contribution of the ImmuKnow assay for longitudinal individualized cellular immune function monitoring of patients following HCT. Further studies are necessary in order to establish the optimal practice for utilizing the assay for this purpose.

Immune functional assay for immunosuppressive management in post-transplant malignancy

Immunosuppression management in post-transplant malignancy is challenging because of a lack of objective immunologic assessment tools. The ImmuKnow assay measures the ATP level from CD4 T cells, quantifying cell-mediated immunity and providing an insight into the immune status of transplant recipients. Its potential use in patients with post-transplant de novo malignancy was evaluated. Thirteen adult transplant patients with de novo malignancy were divided into survivors (n = 9) and non-survivors (n = 4) after malignancy treatment. Tacrolimus and the ImmuKnow levels were monitored before, during, and after malignancy treatment. The ImmuKnow level in non-survivors group was significantly lower before and after malignancy treatment compared to survivors group (p = 0.013 and 0.0014 respectively). In survivor group, the ImmuKnow level was significantly decreased during malignancy treatment (p = 0.019) but recovered to the initial level after the treatment. However, in non-survivor group, the ImmuKnow level remained suppressed throughout the observed period despite a reduction in immunosuppressive drug levels. The ImmuKnow assay can be an objective means evaluating immune status of patients with de novo malignancy. The ImmuKnow assay can express the degree of immune suppression induced by chemotherapeutic or radiation therapy and may be a useful tool in optimizing the timing of re-introduction of immunosuppression after malignancy treatment.

Individualized Immune Monitoring of Cardiac Transplant Recipients by Noninvasive Longitudinal Cellular Immunity Tests

Common immunosuppression strategies after heart transplantation (HTx) are based on accepted target drug levels, disregarding that drug levels do not correlate with the individual patient's pharmacokinetics or with the actual immunosuppressive drug effect on the patient. The Immuknow assay is used for immune monitoring and management of organ transplant recipients. This study evaluated the Immuknow assay for longitudinal immune monitoring of HTx patients throughout various clinical settings. The functional immune response as measured by the Immuknow assay was determined in 327 samples collected from 50 HTx patients at the Rabin Medical Center and was analyzed together with common clinical parameters. The median Immuknow levels measured throughout the infection episodes and the episodes of biopsy-proven acute rejection were 129 and 619 ng ATP/mL, respectively. These values were significantly dissimilar to the median Immuknow level measured during clinical quiescence, which was 351 ng ATP/mL (P<0.05). Calcineurin inhibitors drug-level measurements did not provide a reliable depiction of the patients' immune function, because the median deviation from the recommended drug trough levels range was significantly higher than the median deviation of Immuknow levels from their expected immune response zones. Longitudinal monitoring of Immuknow levels through serial testing proved to be a reliable method for individual patient immune management. The Immuknow assay reliably reflects the cellular immune function of HTx patients, thereby supporting the immune monitoring and management of these patients. Serial longitudinal Immuknow monitoring allows immune management of therapy according to the individual patient's immune status.

Post‐transplant lymphoproliferative disease in pediatric lung transplant recipients: Recent advances in monitoring

To investigate the clinical validity of newer diagnostic tests such as monitoring of EBVqPCR and lymphocyte function assay ImmuKnow in helping to diagnose PTLD in pediatric lung transplant recipients. Single-center, retrospective case-control study. CsA trough levels, EBVqPCR and ImmuKnow (Cyclex Inc., Columbia, MD, USA) levels were measured serially as part of routine care. Re-transplant patients and patients who did not reach 12 months post-transplant at the time of analysis were excluded. Twenty-seven patients met the inclusion criteria. The study group consisted of seven patients who developed PTLD, five of which were EBV- recipients who received EBV+ lungs. The rest of the eligible patients served as controls. Median time to develop PTLD was 273 days (range: 166-343). One, two, three, six, and nine months after transplant, mean (+/-s.d.) CsA trough whole blood levels (ng/mL) were not different between the two groups: 378 +/- 38, 390 +/- 52, 402 +/- 89, 359 +/- 42, and 342 +/- 115, vs. 416 +/- 105, 347 +/- 64, 337 +/- 78, 333 +/- 86, and 281 +/- 54 [PTLD vs. no-PTLD, respectively (p > 0.05 for all time points)]. Mean (+/-s.d.) EBVqPCR levels (copies/mL) measured at three, six, and nine months post-transplant were significantly elevated in PTLD group compared to no-PTLD group: 84 +/- 99, 3384 +/- 7428 and 839 +/- 1444 vs. 9 +/- 26, 8 +/- 36 and 32 +/- 136, respectively (p < 0.05 for all time points). Mean (+/-s.d.) ImmuKnow levels (ATP ng/mL) at three, six, and nine months post-transplant were significantly lower in the PTLD group when compared with no-PTLD group: 144 +/- 67, 137 +/- 110, and 120 +/- 153 vs. 290 +/- 161, 300 +/- 162, and 293 +/- 190, respectively (p < 0.05 for all time points). Close monitoring of EBV viral load by qPCR and the degree of immunosuppression via ImmuKnow may guide physicians to reach the diagnosis of PTLD early.

Cylex ImmuKnow Assay Levels Are Lower in Lung Transplant Recipients With Infection

Current monitoring systems of immunosuppression in solid-organ transplant recipients are typically focused on prevention of clinical toxicities of immunosuppressive drugs. Unfortunately, these strategies are often not tailored to the individual and do not determine the optimal level of immunosuppression for these patients. Recently, the Cylex Immune Cell Function Assay (ImmuKnow; Cylex, Inc., Columbia, MD) was approved by the U.S. Food and Drug Administration (FDA) to measure global immune response in solid-organ transplant patients receiving immunosuppressive therapy. We sought to identify the level of functional immunity as measured by the ImmuKnow assay in lung transplant recipients and to correlate these values with the dose and trough levels of immunosuppression as well as other clinical parameters in lung transplant recipients. We assessed the functional immune response by the ImmuKnow assay in 143 sequential blood samples from 57 lung transplant recipients from Loyola University Medical Center. The average ImmuKnow assay in stable lung transplant recipients was 244 +/- 138 adenosine triphosphate (ATP) ng/ml and the median level was 236 ATP ng/ml (range 5 to 669 ATP ng/ml), approximately 703 +/- 695 days after lung transplantation. There was no correlation between ImmuKnow levels and tacrolimus trough levels. Stepwise multiple regression analysis identified African American race as an independent predictor of ImmuKnow assay levels when age, gender and underlying diagnosis were taken into account (p < 0.04). Fifteen infected lung transplant recipients had a lower ImmuKnow level at the time of their infections as compared with stable lung transplant recipients (111 +/- 83 vs 283 +/- 143 ATP ng/ml, respectively, p = 0.0001). Sixteen of the remaining 42 patients had low ImmuKnow assay values (<225 ATP ng/ml), but did not have active infection. There were only 2 patients with acute rejection of Grade A1 in this cohort. There were no identifiable associations of the ImmuKnow level with either acute rejection episode. The Cylex ImmuKnow assay levels were lower in infected lung transplant recipients compared with non-infected recipients and increased with treatment of these infections. It remains unclear whether the ImmuKnow assay reflects over-immunosuppressed individuals at risk of infection or bone marrow suppression by infectious agents. Further investigation will determine the role of the ImmuKnow assay in tailoring immunosuppression in lung transplant recipients.

251: Importance of Tricuspid Annuloplasty at the Time of Heart Transplantation

had 2 episodes of CMV infection despite prophylactic Gancyclovir and 3 had recurrent respiratory tract infections), 5 experienced biopsy proven rejection and 3 pts were started on a CNI free protocol due to CNI side effects. The contribution of the Immuknow assay measurement was assessed. Results: Mean Immuknow levels were low in the infected pts (235) and high in the pts with rejection (575). During the change to CNI free protocol, mean Immuknow levels were appropriate (426). In the fungal infected pts, the immunosuppressive treatment was diminished to low doses of steroids, lower trough CNI levels, omitting mycophenolate moefetil. The lower Immuknow levels encouraged us to continue the low potency immunosuppression. In the CMV and the respiratory tract infected pts, the Immuknow levels were low despite CNI therapeutic drug levels. Their immunosuppressive regimen was changed to a less potent one. In the pts with rejection, steroids were added and MMF was changed to everolimus. The appropriate Immuknow levels during the change to the CNI free protocol reflect the uneventful protocol change. Conclusions: Monitoring the Immuknow levels is a valuable and simple tool for immunosuppressive therapy monitoring. It helps decision making in complex situations allowing therapeutic changes that can favorably affect the outcome of HTx recipients.

Monitoring Immune Function by the Cylex Immuknow Assay in Pediatric HSCT Patients.

A observational analysis of immune function in six pediatric HSCT patients (3 allogeneic/3 autologous) ranging in age from 3 to 16 years (median = 8.6) was conducted at Children's Hospital; Louisiana State University Health Sciences Center using ImmuKnow®, an FDA cleared assay for assessing T cell activation. This whole blood assay measures the immune function of CD4+ T-helper cells by stimulating cells with phytohemagglutinin (PHA), selecting CD4+ cells with antibody coated magnetic particles and detecting ATP by bioluminescence, an indicator of activation. The immunknow assay was obtained weekly post-HSCT. On average, the ImmuKnow value for the allogeneic patients was 109 ng/mL ATP with a median value of 63 ng/mL ATP. In two of the three allopatients, a rapid and significant rise in immuknow levels correlated with clinical complications. One ALL patient, given an allogeneic PBSCT from a HLA-ID sibling, developed Veno-occlusive disease (VOD) of the liver, 18 days post BMT with a corresponding spike in the ImmuKnow level to 500 ng/mL ATP. Following treatment with defibrotide, the VOD resolved and ImmuKnow ATP levels decreased, stabilizing between 100–150 ng/mL ATP. A second allopatient showed dramatic rise in immuknow level at day 18 post transplant to 480ng/mL ATP that corresponded with gross hematuria secondary to urethritis. In autologous BMT patients, the average ImmuKnow value was 133 ng/mL ATP with a median value of 70 ng/mL ATP. Clinical correlations were less clear in this small sample of patients with fewer test values. ImmuKnow has previously been demonstrated to have clinical utility for managing immunosuppressive therapies in solid-organ transplantation (SOT)1 The range of immune function values in pediatric patients has previously been shown to be significantly lower than adult ranges.2 Our data suggest that this assay may also be useful as a marker for the development of complications post-HSCT. Additional studies are in progress to confirm these early findings that suggest that routine immune function monitoring is of clinical value in the allogeneic HSCT recipients.