|MP 2.04.56||Immune Cell Function Assay|
|Original Policy Date
|Last Review Status/Date
Reviewed with literature search/11:2014
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Our medical policies are designed for informational purposes only and are not an authorization, or an explanation of benefits, or a contract. Receipt of benefits is subject to satisfaction of all terms and conditions of the coverage. Medical technology is constantly changing, and we reserve the right to review and update our policies periodically.
Currently, immunosuppression is determined by testing for clinical toxicity (eg, 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 levels of an individual immunosuppressant drug may not reflect the cumulative effect of other concomitant immunosuppressants. The main value of TDM is the avoidance of toxic levels and monitoring patient compliance. Further, the appropriate level of immunosuppression may vary from person to person. Individual immune profiles, such as an immune cell function assay, could support clinical decision making and help to manage the risk of infection from excess immunosuppression and the risk of rejection from inadequate immunosuppression in immunosuppressed patients.
ImmuKnow® measures the concentration of ATP in whole blood after a 15- to 18-hour incubation with the mitogenic stimulant, phytohemagglutinin. In cells that respond to stimulation, increased ATP synthesis occurs during incubation. Concurrently, whole blood is incubated in the absence of stimulant for the purpose of assessing basal ATP activity. CD4+ T lymphocytes are immunoselected from both samples using anti-CD4 monoclonal antibody-coated magnetic particles. After washing the selected CD4+ 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.
ImmuKnow® (Cylex, recently acquired by Viracor-IBT Laboratories Inc., Lee’s Summit, MO) is an immune cell function assay cleared for marketing by FDA in April 2002 to detect cell-mediated immunity (CMI) in an immunosuppressed patient population.
In April 2002, Cylex obtained 510(k) clearance from FDA to market the Immune Cell Function Assay based on substantial equivalence to 2 flow cytometry reagents (“predicate devices”) manufactured by Becton Dickinson, the TriTestTM CD4 FITC/CD8 PE/CD3 PerCP Reagent and the MultiTestTM CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC Reagent. These reagents are used to determine CD4+ Tlymphocyte counts in immunocompromised patients. The FDA-indicated use of the Immune Cell Function Assay is for the detection of CMI in an immunosuppressed population. A subsequent 510(k) marketing clearance for a device modification was issued by FDA for this assay in 2010. There were no changes to the indications or intended use.(1)
Use of the immune cell function assay to monitor and predict immune function after solid organ transplantation is considered investigational.
Use of the immune cell function assay to monitor and predict immune function after hematopoietic stem cell transplantation is considered investigational.
Use of the immune cell function assay for all other indications is considered investigational.
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, laboratory websites showed one or both of the following codes being used:
86353: Lymphocyte transformation, mitogen (phytomitogen) or antigen induced blastogenesis
82397: Chemiluminescent assay
BlueCard/National Account Issues
State or federal mandates (e.g., FEP) may dictate that all FDA-approved devices, drugs or biologics may not be considered investigational and thus these devices may be assessed only on the basis of their medical necessity.
This policy was created in August 2009 and has been updated annually. The most recent literature search was performed for the period through October 17, 2014.
The ImmuKnow® (Cylex) 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/retest reliability; (2) clinical validity including sensitivity, specificity, and positive and negative predictive value in appropriate populations of patients; and (3) clinical utility, ie, demonstration that the information from the diagnostic test results in improved health outcomes.
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 2 methods in a population of patients who are suspected of having a disease but not all have the disease/disorder. In addition, 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.
Randomized Controlled Trials
There are no published randomized controlled trials (RCTs) that compare immune cell function assays with current methods of assessing immune status. The majority of published studies of the ImmuKnow® assay are observational studies that correlate adenosine triphosphate (ATP) levels with clinical status. Some include additional analyses of the performance characteristics of the test. Systematic reviews of these observational studies have also been performed. No published trials have assessed the clinical utility of the immune cell function assays.
Ling et al (2012) performed a systematic review and meta-analysis of studies published to July 2011 to assess the efficacy of ImmuKnow® for identifying risks of infection and rejection in adult transplant recipients.(2) Nine studies published between 2008 and 2011 met inclusion criteria. Meta-analysis of these 9 studies incorporated 2458 samples from transplant recipients, including 172 samples from patients with infection and 135 samples from patients with rejection. Three studies were of liver transplant recipients, 3 of kidney recipients, and 1 study each of heart, lung, and mixed organ transplant recipients. Pooled estimates of ImmuKnow® performance characteristics for identification of infection risk were: sensitivity of 0.58 (95% confidence interval [CI], 0.52 to 0.64), specificity of 0.69 (95% CI, 0.66 to 0.70), positive likelihood ratio of 2.37 (95% CI, 1.90 to 2.94), negative likelihood ratio of 0.39 (95% CI, 0.16 to 0.70), and diagnostic odds ratio (OR) of 7.41 (95% CI, 3.36 to 16.34). Pooled estimates for ImmuKnow® in identifying risk of rejection were: sensitivity of 0.43 (95% CI, 0.34 to 0.52), specificity of 0.75 (95% CI, 0.72 to 0.78), positive likelihood ratio of 1.30 (95% CI, 0.74 to 2.28), negative likelihood ratio of 0.96 (95% CI, 0.85 to 1.07), and diagnostic OR of 1.19 (95% CI 0.65 to 2.20). Due to significant heterogeneity across studies, review authors conducted subgroup analyses in liver and renal transplant recipients. The liver transplantation group had a pooled sensitivity of 0.85, and the renal transplantation group had a specificity of 0.80, indicating that different types of organ transplanted may be one source of 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 infection risk in either group. Based on the overall findings, current evidence suggests that ImmuKnow® does not have sufficient diagnostic accuracy to identify individuals at risk of infection or rejection. In particular, sensitivity is low, and likelihood ratios close to 1.0 indicate that this test does not alter the probability of specified outcomes to a large degree. Additional studies are needed to clarify the usefulness of this assay for identifying risks of infection and rejection in adult transplant recipients.
Rodrigo et al (2012) conducted a meta-analysis to identify studies (published to March 2012) documenting the use of ImmuKnow® to monitor immune function in adult liver transplant recipients.(3) Five studies analyzed ImmuKnow® performance in infection (total N=651), and 5 in acute rejection (total N=543). Two (of 5) studies also were included in the previously discussed systematic review by Ling et al. Pooled sensitivity, specificity, positive likelihood ratio, diagnostic OR, and mean (SD) area under the summary receiver operating characteristic (ROC) curve for infection were 0.84 (95% CI, 0.78 to 0.88), 0.75 (95% CI, 0.71 to 0.79), 3.3 (95% CI, 2.8 to 4.0), 14.6 (95% CI, 9.6 to 22.3), and 0.824 (0.034), respectively. Pooled estimates for acute rejection were 0.66 (95% CI, 0.55 to 0.75), 0.80 (95% CI, 0.76 to 0.84), 3.4 (95% CI, 2.4 to 4.7), 8.8 (95% CI, 3.1 to 24.8), and 0.835 (0.060), respectively. Heterogeneity was low for infection and high for acute rejection studies. Based on these findings, ImmuKnow® could be considered a valid tool to assess infection risk in adult liver transplant recipients. However, due to significant heterogeneity across studies, conclusions about prediction of rejection risk with ImmuKnow® are limited.
Kowalski et al published a manufacturer-supported meta-analysis in 2006.(4) ImmuKnow® was assessed in 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 across 10 U.S. centers. Because only 5% of patients with ATP production between 130 and 450 ng/mL demonstrated adverse events (either infection or rejection), the authors proposed this as the target range for ATP production 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 cited in the product insert (described next). Further, a 2005 manufacturer-supported study5 of 37 stable pediatric kidney transplant recipients (mean age, 11.1 years) suggested that in children younger than 12 years of age, risk intervals are defined by ATP production greater than 395 ng/mL for rejection and less than 175 ng/mL for infection.
Correlation of ATP levels with clinical status
In its application for approval of ImmuKnow® by the U.S. Food and Drug Administration (FDA), Cylex submitted results from a multicenter study of 44 healthy adults and 78 transplant recipients.(6) This study was expanded to include 115 apparently healthy adults and 127 solid organ transplant recipients (59% kidney, 34% liver, 2% pancreas, 5% simultaneous kidney and pancreas). Immunosuppressive therapies were not limited and included muromonab (lymphocyte-depleting antibody, OKT3), antithymocyte globulin, calcineurin inhibitors (eg, cyclosporine, tacrolimus), steroids, and mycophenolate mofetil, a purine synthesis inhibitor. ImmuKnow® was performed less than 1 month to greater than 4 years after transplant. Additional details of testing were not specified. Ninety-two percent of transplant recipients had CD4+ ATP levels less than 525 ng/mL, and 94% of apparently healthy controls had CD4+ ATP values greater than 225 ng/mL. The authors concluded that this defines 3 zones of immune response: ATP production 225 ng/mL or less indicates that the patient’s circulating immune cells are showing a low response to phytohemagglutinin (PHA) stimulation and suggests that the patient may be at increased risk of infection; ATP production 525 ng/mL or greater 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 production (ie, between 225 and 525 ng/mL) represents a proposed ideal response to PHA stimulation. ATP production was not correlated with CD4+ T-cell count.
A 2005 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 to 190 days posttransplant.(7) In 8 patients successfully tapered from tacrolimus, 70% of ATP production clustered in the low range (less than 225 ng/mL), 25% of ATP production in the moderate range, and 5% in the high range. Incidence of infection was not reported. Twelve unstable transplant recipients (requiring addition of corticosteroid or OKT3) showed ATP production of 30% in the low range, 43% in the moderate range, and 27% in the high range. This study is often described as using ImmuKnow® results to guide tacrolimus dosing. However,
adjustments to the tapering protocol were determined by histologic examination of biopsy results and correlated with ATP production, as described for the unstable group.
Several studies have found no association between ATP production as determined by ImmuKnow® and outcomes in pediatric solid organ transplant recipients. Rossano et al (2009) studied 83 pediatric patients (median age, 4.9 years) undergoing heart transplant.(8) ImmuKnow® was performed at routine follow-up visits from 3 months to more than 5 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 production as measured by ImmuKnow® was detected between patients with or without acute rejection or with or without infection. Further, the manufacturer’s reported risk ranges for rejection (ATP production ≥525 ng/mL) or infection (ATP production ≤225 ng/mL) were not predictive of rejection or infection, respectively. As noted, however, it may be that pediatric patients’ risks for posttransplant infection and rejection correspond to different ATP production. Subsequent retrospective studies by Wong et al,(9) Ryan et al,(10) and Wozniak et al(11) 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.(10)
Torío et al (2011) 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 polymerase chain reaction [PCR]; n=24), or rejection (biopsy-proven acute rejection; n=35).(12) Healthy blood donors served as controls (n=108). Immunosuppressive regimens included pretransplant basiliximab (an interleukin-2 receptor inhibitor) or antithymocyte globulin and posttransplant tacrolimus, mycophenolate mofetil, and corticosteroid, or calcineurin inhibitors. Mean (SD) ATP production 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).
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.(13) In this study, ATP levels in 5 patients who developed viral infections in the early post-transplantation period (<50 days) were within normal limits. Methodologic limitations prevented any conclusion about the association of ATP levels with infections in 8 patients in the late posttransplantation 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.(14) In this study, mean (SE) ATP production in patients with biopsy-proven rejection (389  ng/mL) and borderline/clinical rejection (254  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  ng/mL) and other (345  ng/mL) infections were not statistically lower compared with ATP production in patients without infection (not reported).
Two studies have examined ATP production in adult heart transplant recipients. Gupta et al (2008) studied 125 adult heart transplant recipients, most of whom underwent ImmuKnow® testing more than 1 year posttransplant.(15) There was no apparent association between ATP production and rejection (n=3). For 7 patients who developed infection, median ATP production was 267 ng/mL and did not differ statistically from median ATP production in 104 patients who did not develop infection (282 ng/mL). There was a significant correlation between ATP production and white blood cell count but not between ATP production and absolute lymphocyte count, suggesting that nonlymphocytes also may influence ATP response. This idea is supported by a 1994 study of CD4+ T-cell responsiveness to 3 stimulants
(including PHA) in HIV-positive patients.(16) The authors suggested that assays performed in clinical laboratories should profile immunoregulatory cytokines (eg, interleukin-2), which modulate the complex interplay between cellular and humoral immune mechanisms.
Israeli et al (2010) correlated ImmuKnow® results with clinical status in 50 immunosuppressed heart transplant recipients (median age, 58.5 years).(17) Median ATP value of 280 blood samples collected from patients during clinical quiescence (ie, good clinical status with normal heart function) was 351 ng/mL. ATP values were within the manufacturer’s “moderate” range of immune function (225-525 ng/mL) in 176
(63%) of these samples. 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). Median ATP value of 19 blood samples collected during episodes of fungal or bacterial infection (ie, requiring hospitalization for intravenous antimicrobial therapy) was 129 ng/mL, a statistically significant difference from the value during clinical quiescence (p<0.05). Although 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), blood samples were drawn during the adverse event rather than before.
Cabrera et al (2009) assessed the ability of ImmuKnow® to differentiate between acute cellular rejection (ACR) and recurrent hepatitis C virus (HCV) infection in 42 adult patients who had HCV-related end-stage liver disease as the indication for liver transplantation.(18) 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 PCR, was not used to diagnose HCV. ImmuKnow® was performed with blood collected before biopsy, and biopsy samples were interpreted by histopathologists blinded to ImmuKnow® results. Median ATP value in 12 patients diagnosed with ACR was 283.3 (range, 241.1-423.0), and 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). 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, although 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).
Role of ATP Production in Monitoring Immunosuppressive Therapy
Several single-center retrospective studies have assessed the use of ImmuKnow® to guide immunosuppressive therapy in both adult and pediatric solid organ transplant recipients.(19-22) These studies demonstrate that this approach may help in more individualized immunosuppression across multiple patient groups. For example, in a 2012 Spanish series by Serrano et al in 40 stable pediatric liver transplant recipients, ATP values among patients with monotherapy (cyclosporin A or tacrolimus) were significantly higher than in patients with double immunosuppressive therapy using either cyclosporin A or tacrolimus and mycophenolate mofetil (p=0.005).(20) On the other hand, a 2013 Chinese series by Zhou et al in 259 adult kidney transplant recipients showed ATP values significantly higher in the conventional
group (receiving immunosuppressive triple therapy) compared with the group receiving monotherapy (alemtuzumab depletion) at 180 days after transplantation (p<0.001).(22) Further prospective studies in larger patient populations using multiple time point measurements of ATP production are required to evaluate the utility of ImmuKnow® for monitoring immunosuppression and improving net health outcomes
post-solid organ transplantation.
Test Performance Characteristics
A smaller number of studies provide some evidence on the performance characteristics of the test, by providing data on sensitivity, specificity, predictive value, and/or area under the ROC curve (AUC). Other studies provide analogous information in the form of an OR for the development of infection or rejection. These studies are discussed next.
Reinsmoen et al (2008) studied 126 kidney transplant recipients to determine whether pretransplant immune parameters (ATP production, as well as HLA mismatch, HLA-specific antibodies, and interferongamma precursor frequencies to donor or third-party cells) were associated with posttransplant early acute rejection, unstable creatinine course, and poor graft outcome.(23) Mean (SD) pretransplant ATP production in recipients who had no clinical reason for a biopsy was significantly lower than those in recipients who had biopsy-proven acute rejection at any posttransplant time point up to 36 months (285.3 [143.2] vs 414.3 [138.5] ng/mL, respectively). Recipients who underwent biopsy but had no diagnosis of acute cellular or antibody-mediated rejection had an intermediate value of 333.7 (156.3) ng/mL. Mean (SD) pretransplant ATP production 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, respectively). Post hoc analysis using a cutoff ATP production of 375 ng/mL revealed that recipients with pretransplant ATP greater than 375 ng/mL were significantly more likely to experience acute rejection (OR, 3.67; 95% CI, 1.195 to 11.201). Immune parameters were not used to guide modifications of the immunosuppression protocol. Graft survival and incidence of infection were not reported in this study.
Serban et al (2009) assessed ImmuKnow® results in 76 kidney transplant recipients (mean age, 50 years) receiving antithymocyte globulin induction and maintenance immunosuppression.(24) ATP values 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 infection requiring hospitalization (median, 110 ng/mL) and 44 stable patients (median, 220 ng/mL; p=0.002). Median ATP value in 9 of 11 patients with rejection (230 ng/mL) did not differ significantly from that observed in stable patients (p not reported). Results of 3 patients whose blood was sampled for ImmuKnow® are unknown. ATP activity did not correlate with the number of CD4+ T cells during the first 5 months posttransplant (r=0.129, p=0.153) but did correlate with the number of neutrophils and total WBCs within the first 3 months posttransplant (r>0.4, p<0.001). Because of substantial myeloid cell contamination of cells captured by ImmuKnow® in patients with low CD4+ T-cell counts, the 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 value 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=0.017). There was no association between ATP values and development of rejection or infection at any time during the entire 10-month follow-up. As suggested by the authors, in darbepoetin-treated patients, increased ATP activity may be due to myeloid cell mobilization induced by darbepoetin rather than T-cell activation and does not justify increased immunosuppression. The relationship between ImmuKnow® results and infections was further analyzed using ROC analysis. AUC was 0.736, indicating a fair accuracy of ImmuKnow® results for prediction of infection risk. The ATP cutoff value 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 value for increased risk of infection differs from the manufacturer’s cutoff value of 225 ng/mL However, because of the specific effects of antithymocyte globulin induction, results of this study cannot be extrapolated to transplant recipients receiving no induction therapy or receiving induction agents that do not cause vigorous lymphocyte depletion (eg, alemtuzumab, an anti-CD25 monoclonal antibody).
Zhou et al (2011) grouped 259 Chinese kidney transplant recipients (mean [SD] 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 (biopsyproven 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).(25) Posttransplant immunosuppressive regimens included corticosteroids, calcineurin inhibitors, and mycophenolate mofetil. Median ATP production 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 production in the rejection group were higher than in the stable group (615.9 ng/mL; range, 548.8-743.5; p<0.001). ROC analysis was evaluated to determine optimal ATP cutoffs for infection and rejection in this sample. With a cutoff for infection of 238 ng/mL, sensitivity and specificity were 93% and 100%, respectively (AUC=0.991). For rejection, a cutoff of 497 ng/mL maximized sensitivity and specificity at 92% and 94%, respectively (AUC=0.988).
Huskey et al (2011) 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.(26) ImmuKnow® 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, sex, and time of testing posttransplant who had neither infection nor rejection served as controls. Immunosuppressive regimens included prednisone, calcineurin inhibitors, and mycophenolate mofetil. Eighty percent of patients received pretransplant 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 production did not differ between cases (386 ng/mL) and controls (417 ng/mL; p=0.24). A low ATP production (≤225 ng/mL) was not associated with an increased risk of infection (OR=1.34; 95% CI, 0.64 to 2.82; p=0.43). Forty-seven ImmuKnow® assays were performed in 47 patients with subsequent acute rejection and in matched controls. Mean ATP production did not differ between cases (390 ng/mL) nd controls (432 ng/mL; p=0.25). A high ATP production (≥525 ng/mL) was not associated with an increased risk of rejection (OR=1.87; 95% CI, 0.47 to 8.38; p=0.48).
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®(27,28) or have suggested an alternative approach to determining optimal cutoff values.(29,30) 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).(28) The cutoff used for this analysis was 300ng/mL. To optimize ImmuKnow® performance, Quaglia et al (2014)(29) and Wang et al (2014)(30) 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 3.29 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.(30)
Bhorade et al (2008) assessed the relationship between low posttransplant ATP production (≤225 ng/mL) and recent infection in 57 immunosuppressed adult lung transplant recipients.(31) ImmuKnow® assays were performed in 143 patients at routine clinic visits when each patient was on a stable dose of tacrolimus. Fifteen patients developed infections (bacterial or fungal pneumonia, cytomegalovirus [CMV]
infection); 14 of these (93%) had ATP production less than 225 ng/mL at the time of their infections (sensitivity 93%). Among the 42 noninfected patients, 16 (38%) had ATP production less than 225 ng/mL (specificity 62%). Without comparing postinfection ATP production with preinfection ATP production, it is not possible to draw conclusions about whether low ATP production contributed to or resulted from the development of infection. In a 2013 U.S. single-center study on 175 adult lung transplant recipients, Shino et al reported that ImmuKnow® had some predictive ability but was unlikely to be sufficiently accurate for use in clinical care.(32) AUC was relatively low at 0.61. At a cutoff of 525 ng/mL, there was a significant increase in the risk for acute cellular rejection (OR=2.1; 95% CI, 1.1 to 3.8). However, at this cutoff, sensitivity was 35%, with specificity of 82%. When a cutoff of 425 ng/mL was used, sensitivity was 53%, and specificity was 65%.
Husain et al (2009) assessed the correlation of ImmuKnow® results with different types of infections (bacterial, fungal, viral) in 175 adult lung transplant recipients receiving immunosuppression induction with alemtuzumab.(33) 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 values drawn within 1 month before the episode were analyzed. Median ATP value during stability was 175 ng/mL (25th-75th percentile, 97-306 ng/mL). Significantly lower median ATP values were seen in 13 CMV infections (49 ng/mL, p<0.001), 5 infections with other viruses (1 Epstein-Barr virus, 2 rhinovirus, 1 influenza, and 1 parainfluenza; 70 ng/mL, p<0.05), and 14 bacterial pneumonias (92 ng/mL, p=0.002). Median ATP value in fungal disease (85 ng/mL) did not differ significantly from that in stability (p not reported). Four patients who developed invasive pulmonary aspergillosis all had ATP values less than 50 ng/mL. Generalized estimating logistic regression analysis demonstrated an OR of 2.81 (95% CI, 1.48 to 4.98) for increased risk of infection with ATP values less than 100 ng/mL and an OR of 9 (95% CI not reported) with values less than 50 ng/mL. In comparison, a diagnosis of cystic fibrosis yielded an OR of 2.66 (95% CI, 1.26 to 5.63) and CMV mismatch (donor positive, recipient negative) yielded an OR of 2.97 (95% CI, 1.52 to 5.80). Note that all ImmuKnow® values, both during periods of stability and within the month before infectious episodes, fall below the manufacturer’s cutoff for increased risk of infection (225 ng/mL).
A retrospective study by Kobashigawa et al (2010) correlated ImmuKnow® results from 296 adult heart transplant recipients (mean [SD] age, 54.6 [12.8] years) with infection or rejection episodes occurring within 1 month of assay.(34) Assays were performed between 2 weeks and 10 years posttransplant (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. 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 (SD) ATP production was lower in patients with infection (187  ng/mL) compared with controls (280  ng/mL, p<0.001). Ten percent of ATP production less than 200 ng/mL were associated with infection, and 2% of ATP production greater than 200 ng/mL were associated with infection (p<0.001). Mean (SD) ATP production did not differ between patients who developed rejection (327  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).
To assess ImmuKnow®’s ability to differentiate acute cellular rejection from recurrent HCV infection in patients transplanted for HCV-related liver disease, Hashimoto et al conducted a retrospective review of 54 allograft liver transplant recipients who had concomitant ImmuKnow® results available (mean age, 52 years; range, 40-63).(35) 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® 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. Mean (SD) ATP production in acute cellular rejection (365  ng/mL; range, 210-666) was higher compared with normal biopsy (240  ng/mL; range, 142-387; p=0.006). Mean (SD) ATP production in recurrent HCV (152  ng/mL; range, 20-487) was lower than in both acute cellular rejection (p<0.001) and normal biopsy (p=0.019). Mean (SD) ATP production of patients with overlapping features of both acute cellular rejection and recurrent HCV (157  ng/mL; range, 25-355) did not differ statistically from the other groups. Seventy-three percent of patients with acute cellular rejection had ATP production in the manufacturer-defined moderate range. Eighty-eight percent of patients with recurrent HCV had ATP production in the low range (p<0.001). ROC analysis yielded a cutoff level of 220 ng/mL with sensitivity of 89% and specificity of 91% (AUC=0.93; 95% CI, 0.85 to 1.00).
Cheng et al (2011) evaluated the ability of ImmuKnow® to predict recurrence of hepatocellular carcinoma (HCC) in Chinese patients undergoing liver transplantation for HCC.(36) A threshold ATP production of 175 ng/mL was initially determined from 176 assays of 60 patients with HCC (mean [SD] age, 49.8 [8.7] years), 60 (34%) from patients with recurrent HCC posttransplant, and 116 (66%) from stable patients without HCC recurrence, infection, or biopsy-proven rejection. Mean (SD) ATP production in patients with recurrent HCC (137.8 [6.4] ng/mL) were lower compared with those without recurrence (289.2 [133.9] ng/mL, p<0.01). Sensitivity and specificity for the 175 ng/mL threshold value were 83% and 84%, respectively (AUC=0.869). ImmuKnow® was then administered to a second cohort of 92 patients with HCC undergoing liver transplantation (mean [SD] age, 50.1 [10.3] years). Patients were stratified by high immune response (mean ATP production >175 ng/mL) and low immune response (mean ATP production, ≤175 ng/mL). Seventeen (23%) of 73 patients in the high response group and 16 (84%) of 19 patients in the low response group developed HCC recurrence (p<0.001). Mean (SD) ATP production 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 to 16.13) but not overall survival (OR=2.20; 95% CI, 0.56 to 8.65). This study also correlated ImmuKnow® results with clinical status (infection or rejection) among a cohort of the original 60 patients with HCC plus
45 additional patients with nonmalignant 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 (SD) ATP production during infection (145.2 [87.0] ng/mL) and rejection (418.9 [169.5] ng/mL) differed from mean (SD) level during stability (286.6 [143.9] ng/mL, p<0.01 for both comparisons). ROC analysis showed that optimum cutoff for infection was 200 ng/mL with sensitivity of 79% and specificity of
75% (AUC=0.842). Optimum cutoff for rejection was 304 ng/mL with sensitivity of 80% and specificity of 76% (AUC=0.806). Another retrospective study of 87 liver transplant recipients utilized a cutoff level for rejection of 407 ng/mL based on ROC analysis with sensitivity and specificity of 86% and 81%, respectively (AUC=0.869).(37)
Use in Conditions Other Than Solid Organ Transplantation
Hematopoietic Stem-Cell 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 (2010) assessed ATP production in 16 adult patients (mean age, 52 years) with hematologic malignancies (multiple myeloma, B- or T-cell lymphoma, 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.(38) Mean (SD) ATP production on day 5 of G-CSF therapy in 10 patients who survived more than 2 years after mobilization (673  ng/mL) was higher compared with 5 patients who died within 2 years (282  ng/mL; p=0.014). ROC analysis identified a cutoff of 522 ng/mL for predicting patient survival with sensitivity and specificity of 80% and 100%, respectively (AUC=0.880). Gesundheit et al (2010) examined 170 ATP production collected from 40 patients (median age, 34 years; range, 3-64) after engraftment of allogeneic HSCT for various malignant (acute and chronic myeloid leukemia, acute and chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, myelodysplastic syndrome, ovarian, breast, and testicular cancer) and nonmalignant (severe aplastic anemia, thalassemia major, adrenoleukodystrophy) diseases.(39) ImmuKnow® results were categorized “low” or “normal” according to the manufacturer’s ATP cutoff values and correlated with postengraftment clinical course. Overall survival for the immunocompetent (“normal”) group was 83% (10/12 patients) at 13 months of follow-up. Overall survival for the immunocompromised (“low”) group was 12% (3/25 patients) at 12 months of follow-up.
Natsuda et al (2014)(40) assessed ATP production in 28 patients coinfected with HIV and HCV. These patients were all receiving antiretroviral 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 coinfected group (259 ng/ML [range, 30-613]) were statistically higher compared with the HCV monoinfected 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 coinfected 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 coefficient, p=0.76). The clinical significance of these findings, for either HCV monoinfected liver transplant recipients or HIV/HCV coinfected patients, is unclear.
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.(41) 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).
Ongoing and Unpublished Clinical Trials
An online search of ClinicalTrials.gov identified 1 observational study designed to evaluate ImmuKnow® for measurement of cell-mediated immune response in renal transplant recipients receiving immunosuppressive therapy (NCT01859832). This single-center Canadian study is currently recruiting 22 adult participants with an estimated completion date of December 2013.
Two observational studies in conditions other than solid organ transplantation also are currently listed at ClinicalTrials.gov. Both studies are completed but neither has been published. The first study (NCT00569842) assessed the ability of ImmuKnow® to predict graft-versus-host disease or infection in 45 patients with hematologic malignancies (leukemia, non-Hodgkin lymphoma, chronic lymphocytic leukemia, Hodgkin disease, multiple myeloma, myelodysplastic syndromes, myeloproliferative disorders, aplastic anemia, chronic myelogenous leukemia) who are undergoing allogeneic HSCT. Estimated completion was December 2012. The second study (NCT00618267) compared ATP production of Thelper lymphocytes in 100 patients with multiple sclerosis receiving various immunomodulatory and immunosuppressive therapies. This study was presented at a meeting of the American Academy of Neurology in 2009.
Summary of Evidence
Published studies to date have primarily been small single-center retrospective studies. Studies indicate that adenosine triphosphate (ATP) levels vary among transplant recipients who have evidence of acute infection or transplant rejection, compared with clinically stable patients. Sensitivity and specificity of immune cell function assay have varied in studies reporting these parameters. Based on results from two 2012 systematic reviews of observational studies of ImmuKnow® in adult transplant recipients, estimates of sensitivity range from 52% to 88% for infection and 34% to 75% for rejection. Estimates of specificity, on the other hand, range from 66% to 79% for infection and 72% to 84% for rejection. Given the significant heterogeneity observed across studies, performance characteristics of ImmuKnow® 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 stimulation variably or whether cutoff values require adjustment for various clinical scenarios. prospective trials are needed to better define the predictive ability of ImmuKnow® compared with current methods of assessing immune status.
Clinical utility of ImmuKnow® to impact net health outcome in comparison with current methods of care for solid organ transplant recipients has not been evaluated using prospective trials with multiple time point measurements of ATP production. Thus, it is unknown how current methods of assessing immune status in solid organ transplant recipients, eg, immunosuppressant drug-level monitoring or empiric use of anti-infective agents, might be changed by use of ImmuKnow®. Therefore, the ImmuKnow® cell function assay is considered investigational.
Practice Guidelines and Position Statements
The International Cytomegalovirus Consensus Group of the Transplantation Society published an international consensus statement on the management of cytomegalovirus in solid organ transplant in 2010.(42) Authors stated that “there are no clinical studies demonstrating that management decisions based on immunologic monitoring affect patient outcomes.” Routine immunologic monitoring was not recommended.
International Society of Heart and Lung Transplantation
Guidelines for the care of heart transplant recipients published in 2010 by the International Society of Heart and Lung Transplantation did not include ImmuKnow®.(43)
American Society of Transplantation
In 2006, the American Society of Transplantation published recommendations for the screening, monitoring, and reporting of infectious complications in immunosuppression trials of organ transplant recipients.(44) These recommendations defined relevant infectious complications to be included in the reporting of immunosuppression trials and recommended specific laboratory monitoring and surveillance
methods. The immune cell function assay was not included.
U.S. Preventive Services Task Force Recommendations
Medicare National Coverage
There is no national coverage determination (NCD). In the absence of an NCD, coverage decisions are left to the discretion of local Medicare carriers.
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- Gupta S, Mitchell JD, Markham DW, et al. Utility of the Cylex assay in cardiac transplant recipients. J Heart Lung Transplant. Aug 2008;27(8):817-822. PMID 18656792
- Shearer G, Clerici M. In vitro analysis of cell-mediated immunity: clinical relevance. Clin Chem. November 1, 1994 1994;40(11):2162-2165.
- Israeli M, Ben-Gal T, Yaari V, et al. Individualized immune monitoring of cardiac transplant recipients by noninvasive longitudinal cellular immunity tests. Transplantation. Apr 27 2010;89(8):968-976. PMID 20075792
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- Heikal NM, Bader FM, Martins TB, et al. Immune function surveillance: association with rejection, infection and cardiac allograft vasculopathy. Transplant Proc. Jan-Feb 2013;45(1):376-382. PMID 23267802
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- Te HS, Dasgupta KA, Cao D, et al. Use of immune function test in monitoring immunosuppression in liver transplant recipients. Clin Transplant. Nov-Dec 2012;26(6):826-832. PMID 22554357
- Zhou H, Lin J, Chen S, et al. Use of the ImmuKnow assay to evaluate the effect of alemtuzumab-depleting induction therapy on cell-mediated immune function after renal transplantation. Clin Exp Nephrol. Apr 2013;17(2):304-309. PMID 23053591
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|CPT||86352||Cellular function assay involving stimulation (eg, mitogen or antigen) and detection of biomarker (eg, ATP)|
|ICD-9-CM Diagnosis||Investigational for all applicable codes|
|ICD-10-CM (effective 10/1/15)||Investigational for all applicable diagnoses|
|T86.10 –T86.819; T86.890-T86.899||Complications of transplanted organs and tissue code range limited to solid organ transplants|
|Z94.0 – Z94.4; Z94.83; Z94.89; Z94.9||Transplanted organ and tissue status; kidney, heart, lung, heart & lungs, liver, pancreas, other and unspecified codes|
|ICD-10-PCS (effective 10/1/15)||Not applicable. No ICD procedure codes for laboratory tests.|
Immune Cell Function Assay
Transplantation Immune Cell Function Assay
|08/13/09||Add policy to Medicine section, Pathology/ Laboratory subsection||Policy created with literature search through July 2009; considered investigational|
|09/16/10||Replace policy||Policy updated with literature search, new references 11-14 added, policy statement unchanged.|
|11/10/11||Replace policy||Policy updated with literature search; additional investigational indication added for HSCT and all other indications; references 10, 15-25 added; title changed to “Immune Cell Function Assay.”|
|11/8/12||Replace Policy||Policy updated with literature review through September 2012; two systematic reviews added and summary revised; references reordered; no change in policy statement|
|11/14/13||Replace policy||Policy updated with literature review through October 7, 2013; references 14-17 and 19 added; no change in policy statements|
|11/13/14||Replace policy||Policy updated with literature review through October 17, 2014; references 9-11, 13-14, 27-30, and 40-41 added; references 1 and 43 updated; reference 32 deleted. No change in policy statements.|
|12/30/14||Replace policy- correction only||Kobashigawa citation (reference 34) added back to policy.|