Coverage Policy Manual
Policy #: 2009029
Category: Medicine
Initiated: August 2009
Last Review: January 2024
  Immune Cell Function Assay
Description:
Careful monitoring of lifelong immunosuppression is required to ensure long-term viability of solid organ allografts without incurring increased risk of infection. Monitoring of immunosuppression attempts to balance the dual risks of rejection and infection. It is proposed that individual immune profiles, such as an immune cell function assay, will help assess the immune function of the transplant recipient and individualize immunosuppressive therapy.
 
In current clinical practice, levels of immunosuppression in patients being managed after a solid organ transplant or hematopoietic cell transplantation are determined by testing for clinical toxicity (e.g., leukopenia, renal failure) and by therapeutic drug monitoring (TDM) when available. However, drug levels are not a surrogate for overall drug distribution or efficacy because pharmacokinetics often differ among individuals due to clinical factors such as underlying diagnosis, age, gender and race; circulating drug levels may not reflect the drug concentration in relevant tissues; and serum level of an individual immunosuppressant drug may not reflect the cumulative effect of other concomitant immunosuppressants. The main value of TDM is the avoidance of toxicity. Individual immune profiles, such as an immune cell function assay, could support clinical decision making and help to manage the risk of infection from excessive immunosuppression and the risk of rejection from inadequate immunosuppression.
 
Several commercially available tests of immune cell function have been developed to support clinical decision making.
 
ImmuKnow measures the concentration of adenosine triphosphate (ATP) in whole blood after a 15- to 18-hour incubation with phytohemagglutinin (a mitogenic stimulant). Cells that respond to stimulation show increased ATP synthesis during incubation. Concurrently, whole blood is incubated in the absence of stimulants for the purpose of assessing basal ATP activity. CD4-positive T lymphocytes are immunoselected from both samples using anti-CD4 monoclonal antibody-coated magnetic particles. After washing the selected CD4-positive cells on a magnet tray, a lysis reagent is added to release intracellular ATP. A luminescence reagent added to the released ATP produces light measured by a luminometer, which is proportional to the concentration of ATP. The characterization of the cellular immune response of a specimen is made by comparing the ATP concentration for that specimen with fixed ATP production ranges.
 
Pleximmune measures CD154 expression on T-cytotoxic memory cells in patient’s peripheral blood lymphocytes. CD154 is a marker of inflammatory response. To characterize the risk of rejection, the patient’s inflammatory response to transplant donor cells is expressed as a fraction of the patient’s inflammatory response to third-party cells. This fraction or ratio is called the Immunoreactivity Index (IR). If the donor-induced response exceeds the response to third-party cells, the individual is at increased risk for rejection. Cells are cultured and then analyzed with fluorochrome-stained antibodies to identify the cells expressing CD154. For posttransplant blood samples, an IR greater than 1.1 indicates an increased risk of rejection, and an IR less than 1.1 indicates a decreased risk of rejection. For pretransplant samples, the threshold for IR is 1.23.
 
Regulatory Status
In April 2002, ImmuKnow® (Cylex, acquired by ViraCor-IBT Laboratories), an immune cell function assay, was cleared for marketing by the U.S. Food and Drug Administration (FDA) through the 510(k) process (K013169). The FDA indicated use of ImmuKnow® is for the detection of a cell-mediated immune response in populations undergoing immunosuppressive therapy for an organ transplant.
 
In April 2002, Immune Cell Function Assay (Cylex) was cleared for marketing by FDA through the 510(k) process. The FDA-indicated use of the Immune Cell Function Assay is for the detection of a cell-mediated immune response in an immunosuppressed population. In 2010, a device modification for this assay was cleared for marketing by FDA through the 510(k) (K101911). There were no changes to the indications or intended use (FDA, 2010).
 
In August 2014, Pleximmune™ (Plexision) was approved by FDA through the humanitarian device exemption process (FDA, 2015). The test is intended for use in the pre-transplantation and early and late posttransplantation period in pediatric liver and small bowel transplant patients for the purpose of predicting the risk of transplant rejection within 60 days after transplantation or 60 days after sampling.
 
Effective in 2010, a CPT code has been created for this type of testing:
 
86352: Cellular function assay involving stimulation (e.g., mitogen or antigen) and detection of biomarker (e.g., ATP).
  
Prior to 2010, some independent laboratory web sites indicated one or both of the following codes may be used:
 
86353: Lymphocyte transformation, mitogen (phytomitogen) or antigen induced blastogenesis
 
82397: Chemiluminescent assay
Policy/
Coverage:
Effective September 2013
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
 
Use of the immune cell function assay to monitor and predict immune function after solid organ or hematopoietic stem cell transplantation or for any other indication does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness.
 
For members with contracts without primary coverage criteria, use of the immune cell function assay to monitor and predict immune function after solid organ or hematopoietic stem cell transplantation or for any other indication is considered investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective prior to September 2013
Use of the immune cell function assay to monitor and predict immune function after solid organ transplantation does not meet member benefit Primary Coverage Criteria that there be scientific evidence of effectiveness in improving health outcomes.
 
For contracts without Primary Coverage Criteria, use of the immune cell function assay to monitor and predict immune function after solid organ transplantation is considered investigational.  Investigational services are exclusions in the member benefit certificate of coverage.
Rationale:
The ImmuKnow assay has been examined in clinical trials for its potential use in monitoring immunosuppression medication regimens in solid organ transplant patients.
 
Assessment of a diagnostic technology typically focuses on three analyses: 1) analytic validity including comparison to a “gold-standard” test and test/re-test reliability; 2) clinical validity including sensitivity, specificity, positive and negative predictive value in appropriate populations of patients; and 3) clinical utility that is demonstration that the information
from the diagnostic test results in improved health outcomes.
 
The sensitivity of a test is the ability to detect disease when the disease is present (true positive), while specificity indicates the ability to detect patients who do not have the disease (true negative). Evaluation of clinical validity, therefore, requires independent assessment by two methods in a population of patients suspected of having a disease but not all of whom do have the disease/disorder. Additionally, demonstration of the clinical utility of the ImmuKnow assay would require specifying abnormal levels prior to testing an immunosuppressed patient population, making treatment decisions based on the assay results, and documenting decreased morbidity and/or mortality (such as improved transplant organ survival and/or reduced infectious complications) following these treatment decisions.
 
In support of Cylex’s application for FDA clearance, Kowalski et al. conducted a multi-center study of 155 apparently healthy adults and 127 solid organ transplant recipients (59% kidney, 34% liver, 2% pancreas, 5% simultaneous kidney and pancreas) (Kowalski, 2003). Immunosuppressive therapies among the transplant recipients were not limited and included muromonab (lymphocyte-depleting antibody, OKT3), antithymocyte globulin, calcineurin inhibitors (cyclosporine, tacrolimus), steroids and mycophenolate mofetil, a purine synthesis inhibitor. ImmuKnow assays were performed < 1 month to > 4 years after transplant. Additional details on testing were not specified. Ninety-two percent of the transplant patients had CD4+ ATP levels < 525 ng/ml, while 94% of apparently healthy controls had CD4+ ATP values > 225 ng/ml. The authors conclude that this defines three zones of patients’ immune response: ATP level < 225 ng/ml indicates that the patient’s circulating immune cells are showing a low response to PHA stimulation and suggests that the patient may be at increased risk of infection; ATP level > 525 ng/ml indicates that the patient’s circulating immune cells are showing a strong response to PHA stimulation and suggests that the patient may be at increased risk of transplant rejection; a moderate ATP level (i.e., between 225 and 525 ng/ml) represents a proposed ideal response to PHA stimulation. ATP level was not correlated with CD4+ T-cell count.
 
These transplant recipients were included in a follow-up manufacturer-supported study (Kowalski, 2006). The ImmuKnow assay was completed on a total of 504 immunosuppressed transplant recipients (48% kidney, 30% liver, 17% heart, 5% small bowel) within 30 days after an episode of infection or rejection. Because only 5% of patients with ATP levels between 130 ng/ml and 450 ng/ml demonstrated adverse events (either infection or rejection), the authors propose this as the target range for ATP level in immunosuppressed transplant recipients. Note that this analysis yielded different ATP threshold levels     for infection risk and rejection risk than those developed in the earlier study and cited in the product insert. Further, a 2005 manufacturer-supported study of 37 stable pediatric kidney transplant recipients (mean age = 11.1 years) suggests that in children < 12 years of age, risk intervals are defined by ATP level > 395 ng/ml for rejection and < 175 ng/ml for infection (Hooper, 2005).
 
A manufacturer-supported single-center study assessed 20 small bowel transplant recipients (70% isolated small bowel, 10% multivisceral, 10% modified multivisceral, 10% simultaneous liver, small bowel and pancreas) undergoing tacrolimus tapering per protocol 60-190 days post-transplant (Zeevi, 2005). Among 8 patients successfully tapered from tacrolimus, 70% of ATP levels clustered in the low range (< 225 ng/ml), with 25% of ATP levels occurring in the moderate range and 5% occurring in the strong range. Incidence of infection was not reported. Twelve unstable transplant recipients (requiring addition of steroid or OKT3) showed ATP levels with 30% in the low range, 43% in the moderate range, and 27% in the strong range. This study is often described as using ImmuKnow assay results to guide tacrolimus dosing. However, adjustments to the tapering protocol were determined primarily by histological examination of biopsy results which correlated with ATP levels as described for the unstable group.
 
The relationship between low post-transplant ATP levels (< 225 ng/ml) and recent infection in 57 immunosuppressed adult lung transplant recipients was assessed by Bhorade et al.  One hundred forty-three ImmuKnow assays were performed at routine clinic visits when each patient was on a stable dose of tacrolimus. Fifteen patients developed infections (bacterial or fungal pneumonia, cytomegalovirus infection); 14 of these (93%) had ATP levels < 225 ng/ml at the time of their infections. Among the 42 non-infected patients, 16 (38%) had ATP levels < 225 ng/ml. Without comparing post-infection ATP levels with pre-infection ATP levels, it is not possible to draw conclusions about whether a low ATP level contributes to or results from the development of infection.
 
Of note in this study was the finding that “African American race was an independent predictor of decreased ImmuKnow assay levels that remained significant with stepwise multiple regression analysis. Interestingly, African American lung transplant recipients received higher doses of tacrolimus that led to similar tacrolimus trough levels, but had significantly decreased immunoassay levels.” The authors cite data indicating that “African Americans who have undergone renal transplantation require a 37% mean higher dose of tacrolimus to achieve similar blood concentrations” and conclude that African-Americans “may be over-immunosuppressed based on the concurrently obtained ImmuKnow assay.” Because the authors did not report the incidence of infection among African American patients with ATP level < 225 ng/ml, it is not possible to conclude whether these findings are clinically significant.
 
Two studies found no correlation between ATP levels as determined by the ImmuKnow assay and outcomes in cardiac transplant recipients. Rossano et al. studied 83 pediatric patients (median age, 4.9 years) undergoing heart transplant (Rossano, 2009). ImmuKnow assays were performed at routine follow-up visits from three months to over five years after transplant. There were 26 episodes of acute rejection, 20 (77%) of which were cell-mediated, and the remainder were humoral rejection. There were 38 infections. No difference in ATP levels as measured by ImmuKnow assay was detected between patients with or without acute rejection or with or without infection. Further, the manufacturer’s reported risk ranges for rejection (ATP level > 525 ng/ml) or infection (ATP level < 225 ng/ml) were not predictive of rejection or infection respectively. As noted above, however, it may be that pediatric patients’ risks for post-transplant infection and rejection correspond to different ATP levels.
 
Gupta et al. studied 125 adult heart transplant recipients, the majority of whom underwent ImmuKnow assay testing > 1 year post-transplant (Gupta, 2008). There was no apparent correlation between ATP level and rejection (n=3). For seven patients who developed infection, the median ATP level was 267 ng/ml and did not differ from the median ATP level in 104 patients who did not develop infection (282 ng/ml). There was a significant correlation between ATP level and white blood cell count, but not between ATP level and absolute lymphocyte count, suggesting that non-lymphocytes also may influence the ATP response. This idea is supported by a 1994 study (Shearer, 1994) of CD4+ T-cell responsiveness to three stimulants (including phytohemagglutinin) in HIV+ patients. The authors suggest that assays performed in clinical laboratories should profile immunoregulatory cytokines (e.g., interleukin-2) which modulate the complex interplay between cellular and humoral immune mechanisms.
 
The studies cited above report ImmuKnow assay results obtained after transplant. Reinsmoen and colleagues studied 126 kidney transplant recipients to determine whether pre-transplant immune parameters (ATP level as well as human leukocyte antigen (HLA) mismatch, HLA-specific antibodies, and IFN-gamma precursor frequencies to donor or third-party cells) were associated with post-transplant  early acute rejection, unstable creatinine course, and poor graft outcome (Reinsmoen, 2008). The mean pre-transplant ATP level of recipients who had no clinical reason for a biopsy was significantly different from that of recipients who had biopsy-proven AR at any post transplant time point up to 36 months (285.3 ± 143.2 vs. 414.3 ± 138.5 ng/ml). Recipients who underwent biopsy but had no diagnosis of acute cellular or antibody-mediated rejection had an intermediate mean value of 333.7 ± 156.3 ng/ml. Pre-transplant ATP levels were also significantly higher for recipients with early (< 90 days) unstable creatinine levels, a significant predictor of early acute rejection, than for recipients with stable creatinine values (362.8 ± 141.2 vs. 283.4 ± 146.4 ng/ml). Post-hoc analysis using a cutoff ATP level of 375 ng/ml revealed that recipients with pretransplant ATP > 375 ng/ml were significantly more likely to experience acute rejection (OR=3.67, 95% confidence interval, 1.195, 11.201). The immune parameters were not used to guide modifications of the immunosuppression protocol. Graft survival and incidence of infection was not reported in this study.
 
 
In their discussion, Reinsmoen et al. suggest that CD4+ T-cell production of ATP in response to PHA stimulation may depend upon the immunosuppressive medications employed:
“Induction immunosuppression can nonspecifically reduce precursor frequencies; however, not all T cells are depleted equally. T-cells with a memory phenotype [i.e., CD4+] appear to be less   susceptible to depletion than naïve T-cells. Results from studies of alemtuzumab induction therapy have shown that mature T-cells are profoundly depleted, with memory T-cells, B cells, and monocytes depleted to a lesser degree. Further, the recipients in these trials often experienced reversible rejection episodes that were characterized by a predominant monocyte, not lymphocyte, infiltrate. Our results suggest the induction therapy may have had more profound negating effect on the population of cells detected by the ELISPOT [i.e., interferon-gamma producing memory T-cells] but not the CD4+ population of cells detected by the ATP synthesis assay. The association seen between high pre-transplant ATP levels and acute rejection, as seen in this study, may be immunosuppression protocol dependent. These results suggest the relevance of the immune parameters studied may be influenced by the immunosuppression protocol employed.”
 
The American Society of Transplantation (AST) has published recommendations for the screening, monitoring and reporting of infectious complications in immunosuppression trials of organ transplant recipients (Humar,2009).  These recommendations define relevant infectious complications to be included in the reporting of immunosuppression trials and recommend specific laboratory monitoring and surveillance methods. The immune cell function assay is not included in these recommendations.
 
Clinicaltrials.gov lists five studies assessing the ImmuKnow assay’s ability to predict or monitor the development of infection or rejection in solid organ and hematopoietic stem cell transplant recipients  and in multiple sclerosis patients. None of the studies include modifications of immunosuppressive medication regimens based on ATP level.
 
Without clinical trials demonstrating improved patient outcomes, specifically, reduced incidence of infection, rejection and adverse medication-related effects as a direct consequence of ImmuKnow assay results and subsequent treatment, the evidence is insufficient to permit conclusions concerning the effect of this procedure on health outcomes.
 
Future studies are needed to clarify a number of issues:
 
Issues related to analytic validity:
1. Does CD4+ T-cell ATP production in response to PHA stimulation adequately reflect immune cell function?
2. What is the relationship between CD4+ T-cell count and ATP level as recorded by the ImmuKnow assay?
3. Does altered CD4+ T-cell production of ATP precede and contribute to the development of  infection or rejection, or does it result from an episode of infection or rejection?
 
Issues related to clinical validity and utility:
1. Does the type of transplant or the type of immunosuppressive regimen affect the CD4+ T-cell response to PHA stimulation?
2. Does ethnicity affect performance of the ImmuKnow assay?
3. Can reliable cut-off values for infection or rejection risk be identified?
 
 
2012 Update
A search of the MEDLINE database was conducted through August 2012.  There was no new information identified that would prompt a change in the coverage statement. The following is a summary of the identified relevant literature.
 
Correlation of ATP levels with clinical status
Israeli et al. correlated ImmuKnow® assay results with clinical status in 50 immunosuppressed heart transplant recipients (median age 58.5 years) (Israeli, 2010). The median ATP value of 280 blood samples collected from patients during clinical quiescence (i.e., good clinical status with normal heart function) was 351ng/mL. ATP values were within the manufacturer’s “moderate” range of immune function (225-525 ng/mL) in 176 (63%) of these samples. The median ATP value of 22 blood samples collected during episodes of biopsy-proven acute rejection was 619 ng/mL, a statistically significant difference (p<0.05). The median ATP value of 19 blood samples collected during episodes of fungal or bacterial infection (i.e., requiring hospitalization for intravenous antimicrobial therapy) was 129 ng/mL, a statistically significant difference from the value during clinical quiescence (p<0.05). While these ATP values fall within the manufacturer’s defined ranges for increased risk of infection (<225 ng/mL) and increased risk of rejection (>525 ng/mL), the blood samples were drawn during the adverse event rather than before.
 
Cabrera et al. assessed the ability of the ImmuKnow® assay to differentiate between acute cellular rejection (ACR) and recurrent hepatitis C in 42 adult patients who had hepatitis C virus (HCV)-related endstage liver disease as the indication for liver transplant (Cabrera, 2009). All patients had liver enzyme abnormalities post-transplant and underwent liver biopsy to diagnose both ACR and recurrent HCV. The most sensitive indicator of HCV infection, HCV RNA detection by polymerase chain reaction (PCR), was not used to diagnose HCV. The ImmuKnow® assay was performed with blood collected prior to biopsy, and biopsy samples were interpreted by histopathologists blinded to the results of the ImmuKnow® assay. The median ATP value in 12 patients diagnosed with ACR was 283.3 (range: 241.1-423.0), and the median ATP value in 15 patients diagnosed with recurrent HCV was 148.0 (range 33.7-186.0), a statistically significant difference (p<0.001). The median ATP value in 15 patients with mixed biopsy features of both ACR and recurrent HCV, but predominance of neither, was 234.0 (range: 155.3-325.0), a statistically significant difference from both the ACR group (p=0.02) and the recurrent HCV group (p<0.001). Of note, while 100% of patients with recurrent HCV had ATP values within the manufacturer’s range for increased risk of infection (<225 ng/mL), 100% of patients with ACR had ATP values outside of the manufacturer’s cutoff for increased risk of rejection (>525 ng/mL).
 
Torío et al. grouped 227 samples from 116 kidney transplant recipients (mean age 51.2 years, range 19-77) by clinical course: stable (no infectious syndrome or acute rejection episode 1 month before and after immune cell assay; n=168), infection (fever plus at least 1 positive culture or positive PCR; n=24), or rejection (biopsy-proven acute rejection; n=35) (Torio, 2011). Healthy blood donors served as controls (n=108). Immunosuppressive regimens included pre-transplant basiliximab (an interleukin-2 receptor inhibitor) or antithymocyte globulin and post-transplant tacrolimus, mycophenolate mofetil, and corticosteroid, or calcineurin inhibitors. Mean ATP levels in the stable group (375.3 ± 140.1 ng/mL) and in the control group (436.5 ± 112.0 ng/mL) were higher than in the infection group (180.5 ± 55.2 ng/mL; p<0.001 for both comparisons). No difference was observed between the rejection group (332.5 ± 131.7 ng/mL) and the stable group or the control group (p>0.05 for both comparisons).
 
Test performance characteristics
Zhou et al. grouped 259 Chinese kidney transplant recipients (mean age 38.8 ± 12.3 years) by clinical course: stable (no adverse events 7 days before and after immune cell assay; n=174), infection (clinical and imaging evidence of infection within 7 days before or after assay; n=32), rejection (biopsy-proven acute rejection diagnosed within 7 days before or after assay without antirejection therapy; n=16), or methylprednisolone (intravenous methylprednisolone given to treat biopsy-proven acute rejection within 3 days before or after assay; n=33) (Zhou, 2011). Post-transplant immunosuppressive regimens included corticosteroids, calcineurin inhibitors, and mycophenolate mofetil. Median ATP levels in the infection group (116.4 ng/mL, range 66.3–169.2) and the methylprednisolone group (182.3 ng/mL, range 113.6–388.8) were lower than in the stable group (347.7 ng/mL, range 297.9–411.7,p<0.001 for both comparisons). Median ATP levels in the rejection group were higher than in the stable group (615.9 ng/mL, range 548.8–743.5, p<0.001). The ROC analysis was also evaluated to determine optimal ATP cutoff values for infection and rejection in this sample. With an ATP cutoff value for infection of 238 ng/mL, sensitivity and specificity were 92.9% and 100%, respectively (AUC=0.991). For rejection, a cutoff value of 497 ng/mL maximized sensitivity and specificity at 91.5% and 93.8%, respectively (AUC=0.988).
 
A retrospective study by Kobashigawa et al. correlated ImmuKnow® assay results from 296 adult heart transplant recipients (mean age 54.6 ± 12.8 years) with infection or rejection episodes occurring within 1 month of assay (Kobashigawa, 2010).  Assays were performed between 2 weeks and 10 years post-transplant (N=864). Infection was diagnosed by the treating physician and resulted in antibiotic therapy. Rejection was defined as any treated episode of cellular or antibody-mediated rejection, with or without hemodynamic compromise. Heart transplant recipients without infection or rejection served as controls (n=818 assays). All patients received immunosuppression with tacrolimus, mycophenolate mofetil, and corticosteroids, without induction therapy. Oral prednisone bolus and taper was used for asymptomatic rejection, and antithymocyte globulin was used for rejection with hemodynamic compromise. Mean ATP level was lower in patients with infection (187 ± 126 ng/mL) than in controls (280 ± 126 ng/mL, p<0.001). Ten percent of ATP levels less than 200 ng/mL were associated with infection, and 2% of ATP levels greater than 200 ng/mL were associated with infection (p<0.001). Mean ATP levels did not differ between patients who developed rejection (327 ± 175 ng/mL) and controls (p=0.35). The 200 ng/mL cutoff was chosen based on ROC analysis to maximize sensitivity (71%) and specificity (73%; AUC=0.728).
 
Huskey et al. conducted a single-center, retrospective analysis to assess the predictive ability of ImmuKnow® to identify kidney transplant recipients at risk for opportunistic infection or acute rejection when used in routine clinical management (Huskey, 2011).  ImmuKnow® assay results were categorized according to the manufacturer’s ATP cutoff values and correlated with subsequent infection or rejection occurring within 90 days after the assay. Patients matched for age, gender, and time of testing post-transplant who had neither infection nor rejection served as controls. Immunosuppressive regimens included prednisone, calcineurin inhibitors, and mycophenolate mofetil. Eighty percent of patients received pre-transplant antithymocyte globulin. Standard CMV and Pneumocystis carnii prophylaxis was administered. Ninety-four ImmuKnow® assays were performed in 85 patients with subsequent opportunistic infection and in matched controls. Mean ATP levels did not differ between cases (386 ng/mL) and controls (417 ng/mL; p=0.24). A low ATP level (≤225 ng/mL) was not associated with an increased risk of infection (OR 1.34, 95% CI: 0.64, 2.82, p=0.43). Forty-seven ImmuKnow® assays were performed in 47 patients with subsequent acute rejection and in matched controls. Mean ATP levels did not differ between cases (390 ng/mL) and controls (432 ng/mL; p=0.25). A high ATP level (≥525 ng/mL) was not associated with an increased risk of rejection (OR 1.87, 95% CI: 0.47, 8.38, p=0.48).
 
To assess the ImmuKnow® assay’s ability to differentiate acute cellular rejection from recurrent HCV infection among patients transplanted for HCV-related liver disease, Hashimoto et al. conducted a retrospective review of 54 allograft liver transplant recipients who had concomitant ImmuKnow® assay results available (mean age 52 years, range 40-63) (Hashimoto, 2010).  Liver biopsies were performed every 6 months after liver transplantation and when clinically indicated due to elevated liver function tests. Biopsies were read by a pathologist who was blinded to ImmuKnow® assay results. PCR detection of HCV RNA was not used. Immunosuppressive regimens included basiliximab, calcineurin inhibitors, and mycophenolate mofetil. ImmuKnow® assays were collected before biopsy. Results were divided into 4 groups based on biopsy findings: acute cellular rejection (n=11), recurrent HCV (n=26), normal biopsy (n=12), and overlapping features of both acute cellular rejection and recurrent HCV. The mean ATP level in acute cellular rejection (365 ± 130 ng/mL, range 210-666) was higher than in normal biopsy (240 ± 71 ng/mL, range 142-387; p=0.006). The mean ATP level in recurrent HCV (152 ± 100 ng/mL, range 20-487) was lower than in both acute cellular rejection (p<0.001) and normal biopsy (p=0.019). The mean ATP level of patients with overlapping features of both acute cellular rejection and recurrent HCV (157 ± 130 ng/mL, range 25–355) did not differ from the other groups. Seventy-three percent of patients with acute cellular rejection had ATP levels in the manufacturer-defined moderate range. Eighty-eight percent of patients with recurrent HCV had ATP levels in the low range (p<0.001). ROC analysis yielded a cutoff level of 220 ng/mL with sensitivity of 88.5% and specificity of 90.9% (AUC=0.93, 95% CI: 0.85, 1.00).
 
Cheng et al. evaluated the ability of the ImmuKnow® assay to predict recurrence of hepatocellular carcinoma (HCC) in Chinese patients undergoing liver transplantation for HCC (Cheng, 2011).  A threshold ATP level of 175 ng/mL was initially determined from 176 assays of 60 patients with HCC (mean age 49.8 ± 8.7 years), 60 (34%) from patients with recurrent HCC post-transplant, and 116 (66%) from stable patients without HCC recurrence, infection, or biopsy-proven rejection. Mean ATP levels in patients with recurrent HCC (137.8 ± 66.4 ng/mL) were lower than in those without recurrence (289.2 ± 133.9 ng/mL, p<0.01). Sensitivity and specificity for the 175 ng/mL threshold value were 83.3% and 83.6% respectively (AUC=0.869). ImmuKnow® was then administered to a second cohort of 92 patients with HCC undergoing liver transplantation (mean age 50.1 ± 10.3 years). Patients were stratified by high immune response (mean ATP level >175 ng/mL) and low immune response (mean ATP level ≤175 ng/mL). Seventeen of 73 patients (23.3%) in the high response group and 16 of 19 patients (84.2%) in the low response group developed HCC recurrence (p<0.001). Mean ATP levels were 295.3 ± 85.4 ng/mL and 126.6 ± 37.9 ng/mL in the high and low immune response groups, respectively (p<0.001). High immune response was associated with recurrence-free survival (OR 7.28, 95% CI: 3.23, 16.13) but not overall survival (OR 2.20, 95% CI: 0.56, 8.65). This study also correlated ImmuKnow® assay results with clinical status (infection or rejection) among a cohort of the original 60 patients with HCC plus 45 additional patients with non-malignant liver diseases. ImmuKnow® assays were collected during infection (diagnosed by clinical features, positive microbiologic tests, and imaging), biopsy-proven acute or chronic rejection, and stability (defined as good liver function and good general health at least 2 weeks after transplantation, without evidence of infection, rejection, or tumor recurrence). Immunosuppressive regimens were not defined. Rejection episodes were treated with bolus steroids or antithymocyte globulin. Mean ATP levels during infection (145.2 ± 87.0 ng/mL) and rejection (418.9 ± 169.5 ng/mL) differed from the mean level during stability (286.6 ± 143.9 ng/mL, p<0.01 for both comparisons). ROC analysis showed that the optimum cutoff ATP value for infection was 200 ng/mL with sensitivity of 79.2% and specificity of 75.0% (AUC=0.842). The cutoff value for rejection was 304 ng/mL with sensitivity of 79.6% and specificity of 76.4% (AUC=0.806). Another retrospective study of 87 liver transplant recipients (20) utilized a cutoff level for rejection of 407 ng/mL based on ROC analysis with sensitivity and specificity of 85.7% and 80.9%, respectively (AUC=0.869).
 
Use in conditions other than solid organ transplantation
Two studies examined the role of ImmuKnow® in hematopoietic stem cell transplantation (HSCT), one in autologous transplants and one in allogeneic transplants. Manga et al. assessed ATP levels in 16 adult patients (mean age 52 years) with hematologic malignancies (multiple myeloma, B- or T-cell lymphoma, and acute myeloid leukemia) undergoing mobilization with granulocyte-colony stimulating factor (G-CSF) with or without granulocyte-macrophage-colony stimulating factor (GM-CSF) for autologous HSCT (Manga, 2010). Mean ATP level on day 5 of G-CSF therapy in 10 patients who survived more than 2 years after mobilization (673 ± 274 ng/mL) was higher than in 5 patients who died within 2 years (282 ± 194; p=0.014). ROC analysis identified an ATP cutoff value of 522 ng/mL for predicting patient survival with sensitivity and specificity of 0.8 and 1.0, respectively (AUC=0.880). Gesundheit et al. examined 170 ATP levels collected from 40 patients (median age 34 years, range 3-64) following engraftment of allogeneic HSCT for various malignant (acute and chronic myeloid leukemia, acute and chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, myelodysplastic syndrome, and ovarian, breast, and testicular cancer) and non-malignant (severe aplastic anemia, thalassemia major, and adrenoleukodystrophy) diseases (Gesundheit, 2010).  ImmuKnow® assay results were categorized “low” or “normal” according to the manufacturer’s ATP cutoff values and correlated with post-engraftment clinical course. Overall survival for the immunocompetent (“normal”) group was 83% (10 out of 12 patients) at 13 months of follow-up. Overall survival for the immunocompromised (“low”) group was 12% (3 out of 25 patients) at 12 months of follow-up.
 
Practice Guidelines and Position Statements
The International Cytomegalovirus (CMV) Consensus Group of the Transplantation Society published an international consensus statement on the management of CMV in solid organ transplant in 2010 (Kotton, 2010). The authors state that “there are no clinical studies demonstrating that management decisions based on immunologic monitoring affect patient outcomes.” Routine immunologic monitoring is not recommended.
 
Guidelines for the care of heart transplant recipients published in 2010 by The International Society of Heart and Lung Transplantation do not include ImmuKnow®.  Educational guidelines for the management of kidney transplant recipients in the community setting and for infectious diseases in transplant recipients published in 2009 by the American Society of Transplantation (AST) do not include ImmuKnow®.
 
Summary
The studies described above present evidence that ATP levels vary among transplant patients who have evidence of acute infection or transplant rejection, compared to clinically stable patients. The sensitivity and specificity of the test have varied in studies reporting these parameters, and the overall accuracy as measured by ROC analysis ranged from 74-93%. Thus, the performance characteristics of the test have not been conclusively demonstrated. Further, it remains unclear whether different types of organ transplants or different immunosuppressive regimens affect CD4+ T cells’ response to phytohemagglutinin (PHA) stimulation variably or whether cutoff values require adjustment for various clinical scenarios. Prospective trials are needed to better define the predictive ability of the ImmuKnow® assay.
 
The clinical utility of the ImmuKnow® assay to impact net health outcome in comparison to current methods of care for solid organ transplant recipients has not been evaluated. Thus, it is not known how current methods of assessing immune status in solid organ transplant recipients, e.g., immunosuppressant drug-level monitoring or empiric use of anti-infective agents, might be changed by use of the ImmuKnow® assay.
 
2013 Update
A literature search conducted using the MEDLINE database through August 2013 did not reveal any new information that would prompt a change in the coverage statement. Two systematic reviews identified are summarized below.
Ling et al. performed a systematic review and meta-analysis of studies published to July 2011 to assess the efficacy of ImmuKnow® assay in identifying risks of infection and rejection in adult transplant recipients (Ling, 2012). Nine studies published between 2008 and 2011 met the inclusion criteria. The meta-analysis of these 9 studies incorporated 2,458 samples from transplant recipients, with 172 samples from patients with infection and 135 samples from patients with rejection. Three studies were among liver transplant recipients, 3 among kidney recipients, and 1 study each among heart, lung, and mixed organ recipients, respectively. The pooled estimates for the performance characteristics of the ImmuKnow® assay in identification of infection risk were a sensitivity of 0.58 (95% confidence interval [CI]: 0.52-0.64), a specificity of 0.69 (95% CI: 0.66-0.70), a positive likelihood ratio of 2.37 (95% CI: 1.90-2.94), a negative likelihood ratio of 0.39 (95% CI: 0.16-0.70), and a diagnostic odds ratio of 7.41 (95% CI: 3.36-16.34). The pooled estimates for ImmuKnow® assay in identifying risk of rejection were a sensitivity of 0.43 (95% CI: 0.34-0.52), a specificity of 0.75 (95% CI: 0.72-0.78), a positive likelihood ratio of 1.30 (95% CI: 0.74-2.28), a negative likelihood ratio of 0.96 (95% CI: 0.85-1.07), and a diagnostic odds ratio of 1.19 (95% CI: 0.65-2.20). Due to significant heterogeneity across studies, the review authors also conducted subgroup analyses in both liver and renal transplant patients. The subgroup analysis showed that the liver transplantation group had a relatively high pooled sensitivity of 0.85 and the renal transplantation group had a specificity of 0.80, indicating that the different types of organ transplants may be one source of this observed heterogeneity; however, the positive likelihood ratio of the liver group was low and the negative likelihood ratio of the renal group was high, suggesting that it may be inappropriate to use the assay result to identify the risk of infections in either liver or renal transplant recipients. Based on the overall findings, the current evidence suggests that ImmuKnow® assay does not have sufficient diagnostic accuracy to identify individuals at risk of infection or rejection (Ling, 2012). In particular, the sensitivity is low, and the likelihood ratios that are close to 1.0 indicate that this test does not alter the probability of the specified outcomes to a large degree. Additional studies are still needed to clarify the usefulness of this assay for identifying risks of infection and rejection in adult transplant recipients.
 
Rodrigo et al. conducted a meta-analysis to identify studies (published to March 2012) documenting the use of ImmuKnow® assay to monitor immune function in adult liver transplant recipients (Rodrigo, 2012). The authors identified 5 studies to analyze ImmuKnow® assay performance in infection and 5 studies in acute rejection. Two (of 5) studies were also included in the above systematic review by Ling et al. The studies included a total of 651 cases in the infection meta-analysis and 543 cases in the acute rejection meta-analysis. Pooled sensitivity, specificity, positive likelihood ratio, diagnostic odds ratio and area under a summary receiver operating characteristic curve for infection were 0.84 (95% CI: 0.78-0.88), 0.75 (95% CI: 0.71-0.79), 3.3 (95% CI: 2.8-4.0), 14.6 (95% CI: 9.6-22.3), and 0.824 ± 0.034, respectively. The pooled estimates for acute rejection were 0.66 (95% CI: 0.55-0.75), 0.80 (95% CI: 0.76-0.84), 3.4 (95% CI: 2.4-4.7), 8.8 (95% CI: 3.1-24.8) and 0.835 ± 0.060, respectively. Heterogeneity was low for infection and high for acute rejection studies. Based on these findings, the ImmuKnow® assay could be considered a valid tool to know the risk of further infection in adult liver transplant recipients. However, there was significant heterogeneity across studies, which precluded concluding that ImmuKnow® assay identifies liver transplant patients at risk for rejection (Rodrigo, 2012).
 
2014 Update
A literature search conducted through November 2014 did not reveal any new information that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
Pediatric Transplantation
Retrospective studies by Wong et al (Wong, 2014), Ryan et al (Ryan, 2014),and Wozniak et al (Wozniak, 2014), found no association between ATP production and outcomes in pediatric recipients of heart, kidney, or intestinal transplantations, respectively. Ryan et al observed a positive correlation between total peripheral white blood cell (WBC) count and ATP production (r=0.28, p=0.04) and suggested that proportion of activated T cells within submitted samples may provide more useful information (Ryan, 2014).
 
Two retrospective studies of kidney transplant recipients found statistically significant correlations between ATP production and WBC. In a study of 39 patients at a single center in Japan, Nishikawa et al (2014) reported correlation coefficients (R2) of 0.573 (p=0.03) and 0.510 (p=0.02) for associations between WBC and neutrophil counts, respectively (Nishikawa, 2014). In this study, ATP levels in 5 patients who developed viral infections in the early post-transplantation period (<50 days) were within normal limits. Methodological limitations prevented any conclusion about the association of ATP levels with infections in 8 patients in the late post-transplantation period (>120 days). In a study of 306 patients at a single U.S. center, Sageshima et al (2014) reported a correlation coefficients (R2) of 0.264 (p<0.001) for the association between ATP production and WBC (Sageshima, 2014). In this study, mean (SE) ATP production in patients with biopsy-proven rejection (389 [56] ng/ml) and borderline/clinical rejection (254 [41] mg/mL) were not statistically higher compared with ATP production in patients without rejection (not reported). Mean (SE) ATP production in patients with opportunistic (349 [48] ng/mL) and other (345 [27] ng/mL) infections were not statistically lower compared with ATP production in patients without infection (not reported).
 
Kidney Transplantation
Subsequent studies in kidney transplant recipients have demonstrated no association between ATP production and risk of acute rejection or viral infections using manufacturer-recommended cutoffs for ImmuKnow® (Libri, 2013; Myslik, 2014) or have suggested an alternative approach to determining optimal cutoff values (Quaglia, 2014; Wang, 2014). In a prospective cohort study of 55 patients followed for 3 years, Libri et al (2014) observed that ATP production was often lower in patients with acute rejection compared with patients without acute rejection, and was often greater in patients with infection compared with patients without infection. Using labelled cutoffs for ImmuKnow®, AUC was 0.44 (95% CI: 0.18 to 0.71) for acute rejection and 0.37 (95% CI: 0.22 to 0.53) for viral or major respiratory tract infections. In a prospective study of 67 patients undergoing kidney transplant, patients with low preoperative ATP production had statistically fewer rejection episodes than those with high preoperative ATP production (p<0.001) (Myslik, 2014).  The cutoff used for this analysis was 300 ng/mL. To optimize ImmuKnow® performance, Quaglia et al (2014)  and Wang et al (2014) both proposed assessing change in ATP production over time, rather than single values. In a retrospective study of 118 patients, Quaglia et al reported AUC of 0.632 (95% CI: 0.483 to 0.781) for infection risk using a cutoff of –30 ng/mL for the decrease in ATP production from month 1 to month 3. In a prospective study of 140 patients, Wang et al reported AUC of 0.929 for risk of acute rejection using a cutoff of 172.55 ng/mL for the increase in ATP production from “right before” the rejection episode to the occurrence of rejection.
 
HIV
Natsuda et al (2014) (Natsuda, 2014) assessed ATP production in 28 patients co-infected with HIV and HCV. These patients were all receiving anti-retroviral therapy with undetectable viral load in most, and were classified Child-Pugh class A. Results were compared with those of 24 HCV-infected liver transplant recipients and 11 healthy volunteers. Median ATP levels in the HIV/HCV co-infected group (259 ng/ML [range: 30-613]) were statistically higher compared with the HCV mono-infected group (33 ng/mL [range: 6-320]; Mann-Whitney U test, p<0.001) and significantly lower compared with healthy volunteers (446 ng/mL [range: 309-565]; Mann-Whitney U test, p=0.001). In HIV/HCV co-infected patients, ATP production was significantly correlated with CD4+ cell count (Spearman rank correlation, p=0.03) but not with CD4/CD8 ratio (Spearman rank correlation, p=0.76). The clinical significance of these findings, for either HCV mono-infected liver transplant recipients or HIV/HCV co-infected patients, is unclear.
 
Lupus Nephritis
Liu et al (2014) compared ATP production in 22 patients with lupus nephritis and severe infection requiring hospitalization, 74 patients with lupus nephritis and no infection, and 28 healthy controls (Liu, 2014). Mean ATP production was significantly lower in patients with lupus nephritis and severe infection compared with non-infected patients and healthy controls (p<0.01 for both comparisons). Mean ATP production in noninfected LN patients did not differ statistically from that in healthy controls. Using a cutoff of 300 ng/mL, sensitivity and specificity for severe infection were both 77%. Strength of the correlation between ATP production and severe infection (r=–0.040, p<0.001) was less than that between C-reactive protein and severe infection (r=0.962, p<0.001).
   
2015 Update
 
This update includes information relating to the Pleximmune™ test.
 
Analytic Validity of Pleximmune™
U.S. Food and Drug Administration (FDA) documents review precision testing of Pleximmune testing evaluating run-to-run, operator-to-operator, and day-to-day variability (FDA, 2015). All results met the sponsor’s acceptance criteria for an acceptable percentage of the coefficient of variation. No data were presented on the variability of test results within individuals over the short term (representing the same clinical state).
 
Clinical Validity of Pleximmune™
FDA documents describe a clinical validation study of Pleximmune™ FDA, 2015). Among a sample of 33 pre-transplant patients, Pleximmune™ had 57% sensitivity and 89% specificity for identifying rejection. Among a sample of 64 posttransplant patients, Pleximmune™ had 84% sensitivity and 80% specificity for identifying rejection. Almost no details were provided on study validation. A study by Ashokkumar et al evaluated the association of CD154 expression with rejection among pediatric liver transplant patients (Ashokkumar, 2009). It is difficult to determine if the measure of CD154 expression used in this study is the same as the Pleximmune™ test. Using a different threshold value of IR than the current test, IR was associated with the risk of rejection.
 
Clinical Utility of Pleximmune™
We did not identify any studies attempting to document clinical utility, in terms of directly showing improved patient outcomes using Pleximmune™, or using indirect chain of logic.
 
2016 Update
A literature search conducted through November 2016 did not reveal any new published literature that would prompt a change in the coverage statement. The following is a summary of the relevant identified studies.
 
ImmunKnow
Piloni et al (2016) reported on a retrospective cohort study evaluating the immunosuppressive association between over- (ImmuKnow score, corresponding to intracellular ATP, ≤226 ng/mL) versus adequate or undersuppression (ImmuKnow score, >226 ng/mL) in a sample of 61 patients in follow-up for lung transplantation (Piloni, 20160. ImmuKnow testing had been performed at 6-month follow-up for patients who entered the study at the time of transplant (N=28); for other patients, testing was obtained on an as-needed basis because of acute graft dysfunction or suspected immune oversuppression. Being in the immune oversuppression group was associated with higher odds of infection (51 cases of infection/71 ImmuKnow tests vs 25/56; OR=2.754; 95% CI, 1.40 to 5.39; p=0.003). However, given that many patients tested in the as-needed group may have been tested because of suspected immune oversuppression, the risk of bias is very high.
 
Pleximmune
A 2016 study by Ashokkumar et al reported on the preclinical development and validation of an allo-specific CD154+ T-cytotoxic memory cell (TcM) test to predict ACR after liver or intestine transplantation in patients with pediatric liver or lung transplantation (Ashokkummar, 2016). Plexision (manufacturer of Pleximmune) was involved in the study design and assay standardization. A total of 127 patients (120 analyzable samples) were included in the training set (enrolled from 2006 to 2010) and 87 patients (72 analyzable samples) e were included in the validation set (enrolled from 2009 to 2012). The training and test sets differed significantly in terms of organ type composition, with a higher proportion of those in the training set represented by liver or liver/small bowel transplant (eg, 83% liver in training set vs 71% in validation set; p=0.007, for difference between groups). The Immunoreactivity Index (IR) was defined as the ratio of the reaction of donor-induced CD154+ TcM to the reaction exceed those induced by reference peripheral blood leukocytes; a ratio above 1 was considered to indicate an increased risk of rejection. An IR of 1.1 or greater as a cutoff in posttransplant samples was associated with an AUROC curve of 0.878 in the test set (0.791 in the validation set), while a pretransplant IR of 1.23 or greater was an associated with an ROC curve of 0.82 in the training set (0.842 in the validation set).   
 
2017 Update
A literature search conducted through November 2017 did not reveal any new information that would prompt a change in the coverage statement.  
 
2018 Update
A literature search was conducted through November 2018.  There was no new information identified that would prompt a change in the coverage statement.
 
2019 Update
A literature search was conducted through September 2019.  There was no new information identified that would prompt a change in the coverage statement.  The key identified literature is summarized below.
 
Transplantation Society
The International Cytomegalovirus Consensus Group of the Transplantation Society updated its consensus statement on the management of cytomegalovirus in solid organ transplant in 2018 (ICCG, 2018). The statement indicated that “there are no clinical studies demonstrating that management decisions based on immunologic monitoring affect patient outcomes.” Routine immunologic monitoring was not recommended.
 
2021 Update
Annual policy review completed with a literature search using the MEDLINE database through November 2021. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
A prospective cohort study conducted by Xue et al evaluated 216 pediatric patients (mean age, 7 months; range, 3 to 36 months) undergoing liver transplantation from 2 medical centers in China (Xue, 2021). Among the patients, 97.7% (n=211) underwent living donor transplant and the other patients underwent deceased donor transplant. ImmuKnow testing was performed a maximum of 5 days pre-transplantation and weekly from weeks 1 to 4 post-transplantation and once at 8 weeks, 12 weeks, and 24 weeks post-transplantation. Testing was also performed if an episode of infection or rejection occurred. Patients were categorized based on clinical status of stable (clinical, experimental, and imaging examinations without infection or rejection; n=44), infection (signs, symptoms, and imaging consistent for infection and a positive polymerase chain reaction; n=160), and rejection (biopsy-proven acute rejection or elevated liver function tests consistent with rejection; n=12). Immunosuppression regimens included tacrolimus and corticosteroids with or without mycophenolate mofetil. The median pre-transplant ATP level in the full cohort was 193 ng/mL. The median post-transplant ATP levels were significantly lower in the infection group than those in the stable group (137 ng/mL vs. 269 ng/mL, respectively; p<.0001). There was no significant difference between the rejection and stable groups in ATP levels. An ROC curve was generated to determine a reference ATP level for the diagnosis of infection. At an ATP level of 152 ng/mL in patients diagnosed with an infection, the sensitivity and specificity were 57.3% and 95.5%, respectively; the AUC was 0.784 (95% CI, 0.72 to 0.848; p<.0001).
 
Narasimhan et al conducted a retrospective cohort study evaluating effects of the 2-dose SARS-CoV-2 messenger RNA vaccination series (Moderna vs. Pfizer) on humoral response in immunocompromised lung transplant patients through various antibody response measurements using SARS-CoV-2 anti-nucleocapsid protein Immunoglobulin G (IgG) assay (IgGNC), SARS-CoV-2 anti-spike protein Immunoglobulin M (IgM) assay (IgMSP), and SARS-CoV-2 anti-spike protein IgG II assay (IgGSP) (Narasimhan, 2021). As a marker of immunocompetence, CD4-positive T-cell activity was assessed with ImmuKnow testing, measured in 56 of the 73 lung transplant recipients included in the study. Results were interpreted based on manufacturer ATP ranges of low (<225 ng/mL), moderate (226 to 524 ng/mL), or strong (>525 ng/mL). In patients who received the Moderna vaccine series, a positive IgGSP response was demonstrated in 44% (4 out of 9) of patients found to have moderate ImmuKnow values and 50% (1 out of 2) of patients with strong ImmuKnow values. In patients who received the Pfizer vaccine series, a positive IgGSP response was demonstrated in only 18% (3 out of 17) of patients with a moderate ImmuKnow response and no patients (0 out of 6) with strong ImmuKnow levels. The ImmuKnow assay did not give any insight into predicting which patients may have a better antibody response for IgGSP, IgMSP, or IgGNC for either vaccine.
 
In 2019, the American Society of Transplantation Infectious Diseases Community of Practice updated guidelines on post-transplant lymphoproliferative disorders in solid organ transplant (Allen, 2019). A statement indicated: "Simpler rapid assays to measure global and [Epstein-Barr virus] EBV-specific T-cell immunity using commercial ATP release assays (Cyclex ImmuKnow and T-cell Memory) have undergone preliminary evaluation as adjunct markers of [post-transplant lymphoproliferative disorders] PTLD risk when combined with viral load testing in pediatric thoracic transplant recipients but require further validation." Routine immunologic monitoring was not recommended.  
 
2022 Update
Annual policy review completed with a literature search using the MEDLINE database through November 2022. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
In 2018, the International Cytomegalovirus Consensus Group of the Transplantation Society updated its consensus statement on the management of cytomegalovirus in solid organ transplant (Kotton,2018; Kotton, 2010). The statement indicated that “there are no clinical studies demonstrating that management decisions based on immunologic monitoring affect patient outcomes.” Routine immunologic monitoring was not recommended.
 
2023 Update
Annual policy review completed with a literature search using the MEDLINE database through November 2023. No new literature was identified that would prompt a change in the coverage statement. The key identified literature is summarized below.
 
A correlation was found between low ATP levels and infection following a living-donor liver transplantation in pediatric patients (Liu, 2019). The retrospective analysis evaluated 66 patients from a single center in China. The patients were divided into 2 groups: those who were diagnosed with an infection post-transplant (n=28) and those who did not develop an infection (n=38). ImmuKnow testing was performed pre-transplant and at 1 to 4 weeks, 2 months, and 3 months post-transplant. The mean pre-transplant ATP level in the overall cohort was 302.5±195.7 ng/mL. The post-transplant ATP levels were significantly lower in the infection group(188.6±93.5 ng/mL) compared to the non-infection group (424.4±198.1 ng/mL; p<.05). An ROC curve was generated to determine a reference ATP level for the diagnosis of infection. At an ATP level of 200.5 ng/mL in patients diagnosed with an infection, the sensitivity and specificity were 89.5% and 64.3%, respectively; the area under the curve (AUC) was 0.866.
 
ImmuKnow results were assessed for 76 kidney transplant recipients (mean age, 50 years) receiving antithymocyte globulin induction and maintenance immunosuppression (Serban, 2009). ATP levels were assigned to episodes of infection or rejection only if ImmuKnow measurement was performed within 30 days preceding the adverse event. Over a median of 10 months of follow-up, there was a statistically significant difference between ATP activity measured in 15 of 18 patients with an infection requiring hospitalization(median, »110 ng/mL) and 44 stable patients (median, »220 ng/mL; p=.002). Median ATP production for 9 of 11 patients with rejection(230 ng/mL) did not differ significantly from that observed in stable patients (p-value not reported). Results of 3 patients whose blood was sampled for ImmuKnow are unknown. ATP activity did not correlate with the number of CD4-positive T-cells during the first 5months posttransplant (r=0.129; p=.153) but did correlate with the number of neutrophils and total WBCs within the first 3 months posttransplant (r>0.4; p<.001). Because of substantial myeloid cell contamination of cells captured by ImmuKnow in patients with lowCD4-positive T-cell counts, authors concluded that cells of the myeloid lineage substantially contributed to the ATP signal measured by ImmuKnow in these patients. Among 31 patients treated with darbepoetin, median ATP production within the first 2 months posttransplant was approximately 260 ng/mL compared with 160 ng/mL in 38 patients who did not receive darbepoetin (p=.017).There was no association between ATP production and development of rejection or infection at any time during the entire 10-monthfollow-up. As suggested by the authors, in darbepoetin-treated patients, increased ATP activity might be due to myeloid cell mobilization induced by darbepoetin rather than T-cell activation and does not justify increased immunosuppression. The relation between ImmuKnow results and infections was further analyzed using ROC curve analysis. The AUC was 0.736, indicating a fair accuracy of ImmuKnow results for predicting infection risk. The ATP cutoff calculated based on the ROC curve was 165 ng/mL, and corresponding positive and negative predictive values were 0.513 and 0.874, respectively. This cutoff for increased risk of infection differs from the manufacturer’s cutoff of 225 ng/mL. However, because of the specific effects of antithymocyte globulin induction, the results of this study cannot be extrapolated to transplant recipients not receiving induction therapy or receiving induction agents that do not cause vigorous lymphocyte depletion (e.g., alemtuzumab, an anti-CD25 monoclonal antibody).
 
Four studies have examined ATP production in adult heart transplant recipients. The use of ImmuKnow in heart transplant recipients was evaluated with severe systemic infections (Weston, 2020). Patients were followed at the time of scheduled biopsy and weekly with the ImmuKnow assay if diagnosed with a systemic infection. On detection of a systemic infection, maintenance immunosuppression, typically mycophenolate mofetil or azathioprine, was withdrawn and tacrolimus dose was reduced by 50%. Weekly ImmuKnow levels informed further dose reductions of tacrolimus, but the procedure for these reductions was not reported. Maintenance immunosuppression was restarted once the infection was cleared and ImmuKnow levels increased to greater than 225 ng/mL. Thirteen patients had severe systemic infections accounting for 16 total infectious episodes. At the time of the infection, the mean ImmuKnow level was 109 ± 49 ng/mL (from 311 ± 118 ng/mL prior to the diagnosis) and increased to 315 ± 135 ng/mL after the infection cleared (p<.01). The ImmuKnow level during the infection also correlated with the underlying infectious microorganism. Infections caused by a virus, a fungus, or a bacteria had mean ImmuKnow levels of 75 ng/mL, 95.07 ng/mL, and 123.4 ng/mL, respectively. Patients without infections or non‐severe systemic infections served as a control group (n=67). The control group had a mean ImmuKnow level of 294 ± 167 ng/mL. There were 8 episodes of moderate rejection and 6 episodes of severe rejection out of a total of 435 endomyocardial biopsies and 7 episodes of infection in the control group. The mean ImmuKnow level in patients with rejection was 368.7 ng/mL and with infection was 183.3 ng/mL. The study was limited by its single center design and lack of statistical comparisons between patients with severe infections and the control group.
 
The correlation between ImmuKnow results and different types of infections (bacterial, fungal, viral) was assessed in 175 adult lung transplant recipients receiving immunosuppression induction with alemtuzumab (Husain, 2009). Blood samples were collected prospectively as part of routine surveillance in all patients during 2 to 48 months of follow-up. Periods of stability were defined as no infection occurring 1 month before or after the blood draw. For infectious episodes, only ATP levels drawn within 1 month before the episode were analyzed. Median ATP production during stability was 175 ng/mL (25th-75th percentile, 97 to 306 ng/mL). Significantly lower median ATP production levels were seen in 13 cytomegalovirus infections (49 ng/mL; p<.001) and 14 bacterial pneumonias (92ng/mL; p=.002). Median ATP production for fungal disease (85 ng/mL) did not differ significantly from that in stability (p-value not reported). Four patients who developed invasive pulmonary aspergillosis all had ATP levels of less than 50 ng/mL. Generalized estimating logistic regression analysis demonstrated odds of 2.81 (95% CI, 1.48 to 4.98) for increased risk of infection with ATP levels less than 100 ng/mL; moreover, the analysis demonstrated an OR of 9 (95% CI not reported) with values less than 50 ng/mL. In comparison, a diagnosis of cystic fibrosis yielded odds of 2.66 (95% CI, 1.26 to 5.63); cytomegalovirus mismatch (donor positive, recipient negative) yielded an OR of 2.97 (95% CI, 1.52 to 5.80). Note that all ImmuKnow levels, both during periods of stability and within the month before infectious episodes, fell below the manufacturer’s cutoff for increased risk of infection (225 ng/mL).
 
In a 2012 U.S. single-center study on 175 adult lung transplant recipients, it was reported the ImmuKnow test had some predictive ability but was unlikely to be sufficiently accurate for use in clinical care (Shino , 2012). The AUC was relatively low (0.61). At a cutoff of 525 ng/mL, there was a significant increase in the risk for ACR (OR, 2.1; 95% CI, 1.1to 3.8). However, at this cutoff, sensitivity was 35% and specificity was 82%. When a cutoff of 425 ng/mL was used, sensitivity was 53% and specificity was 65%.
 
2024 Update
Annual policy review completed with a literature search using the MEDLINE database through December 2023. No new literature was identified that would prompt a change in the coverage statement.
CPT/HCPCS:
86352Cellular function assay involving stimulation (eg, mitogen or antigen) and detection of biomarker (eg, ATP)
86353Lymphocyte transformation, mitogen (phytomitogen) or antigen induced blastogenesis
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