An immune cell function assay, the ImmuKnow^TM (Cylex^®_, Columbia, MD), was developed for use in the detection of changes in cell mediated immunity (CMI) in individuals undergoing immunosuppressive therapy following solid organ transplant. The assay has also been investigated as a method of identifying individuals at risk for early acute kidney transplant rejection prior to the transplant and for the evaluation of a variety of other conditions such as autologous and allogeneic hematopoietic stem-cell transplant recipients, immunodeficiency diseases and multiple sclerosis.

Investigational and Not Medically Necessary: 

An immune cell function assay is considered investigational and not medically necessary for all indications including, but not limited to:

• For management of organ transplant rejection in an individual undergoing immunosuppressive therapy following solid organ transplant
• For identification of an individual at risk for rejection prior to kidney or any other solid organ transplant
• For management of an individual undergoing autologous or allogeneic hematopoietic stem cell transplant
• For management of an individual with an immunodeficiency disease (e.g. severe combined immunodeficiency disease [SCID])
• For management of an individual with multiple sclerosis

A challenge of transplant management is evaluating the effect of immunosuppressive therapy on the transplant recipient. Too much immunosuppression may result in infection and too little immunosuppression may result in increased risk of organ rejection. An immune cell function assay, the ImmuKnow, has received FDA clearance through the 510(k) process for the detection of CMI response in those undergoing immunosuppressive therapy for organ transplant. This assay measures the concentration of adenosine triphosphate (ATP) released from circulating CD4 cells following a 15-18 hour incubation of a peripheral blood sample with phytohemagglutinin (PHA), a global stimulator of the immune system.

Solid Organ Transplant Management

Kowalski and colleagues (2006) reported on a meta-analysis of 504 solid organ transplant recipients (heart, kidney, kidney-pancreas, liver and small bowel) from 10 U.S. centers in which the Cylex ImmuKnow assay was utilized. Data were collected prospectively during observational studies throughout the United States and combined with cross-sectional data used to support the U.S. Food and Drug Administration (FDA) clearance of the assay. Blood samples were taken from recipients at various times post transplant and compared with clinical course (stable, rejection, infection). In this analysis, 39 biopsy-proven cellular rejections and 66 diagnosed infections occurred. Odds ratios of infection or rejection were calculated based on measured immune response values. Results demonstrated that a recipient with an immune response value of 25 ng/ml ATP was 12 times (95% confidence of 4 to 36) more likely to develop an infection than a recipient with a stronger immune response. Similarly, a recipient with an immune response of 700 ng/ml ATP was 30 times (95% confidence of 8 to 112) more likely to develop a cellular rejection than a recipient with a lower immune response value. The intersection of odds ratio curves for infection and rejection in the moderate immune response zone (280 ng/ml ATP) was noted. This intersection of risk curves provides an immunological target of immune function for solid organ recipients. The authors concluded Cylex ImmuKnow assay has a high negative predictive value and provides a target immunological response zone for minimizing risk and managing individuals to stability.

Cadillo-Chávez and colleagues (2006) evaluated the records of all individuals who received a kidney transplant at a single institution between August 2004 and January 2005 and searched for associations between ATP levels and immunosuppression type, doses, and levels; creatinine levels; white blood cell count; tissue typing; preformed antibodies; as well as ATP levels on infection and rejection, and changes in ATP levels with time. From a total of 64 recipients, 58 had pre and post transplant ATP level determinations. There was no relation found between ATP levels and immunosuppression type, doses, or levels; creatinine levels; white blood cell counts; HLA; and panel-reactive antibody. Individuals with moderate or high pretransplant ATP levels had more rejection episodes (8/10) while those with ATP levels in the low immune response had more infections (6/11). The mean ATP levels for rejection was 423.3 ng/mL versus 268.45 ng/mL for infection and 277.15 ng/mL for no events. Although acute rejections occurred mostly above 300, this was not determined to be significant. Infections were more frequent with ATP under 300 and severe infection (endocarditis, meningitis, peritoneal abscesses, pneumonia, etc.) were more frequent under 200. When pretransplant values were compared with posttransplant values at the second week an increase correlated with rejection, while a decrease did not correlate with the infection. Individuals who received antirejection treatment had a decrease in their ATP levels at five days. Limitations of this study included a small sample size.

Reinsmoen and colleagues (2008) reported on a study aimed at determining whether pretransplant immune parameters were associated with posttransplant early acute rejection, unstable creatinine courses, and poor graft outcome. Immune parameters, including human leukocyte antigen (HLA) mismatch, HLA-specific antibodies, global CD4⁺ cellular response as measured by intracellular adenosine triphosphate (iATP) synthesis, and IFN-gamma precursor frequencies to donor or third-party cells as measured by ELISPOT were determined for a total of 126 kidney recipients treated with a protocol, including rapid discontinuation of prednisone. Results reported included: the donor specific pretransplant parameters of HLA class I mismatches (P=0.04) and total HLA mismatches (P=0.04) with the donor as well as the pretransplant HLA-donor specific antibodies (P=0.002) were associated with biopsy-proven acute rejection. Higher pretransplant iATP levels, a donor nonspecific parameter, were found associated with biopsy proven acute rejection (P=0.04). Pretransplant iATP levels were significantly greater for recipients with early unstable creatinine levels (P=0.01). Recipients with a pretransplant iATP value greater than 375 ng/ml were 3.67 times more likely to experience acute rejection (P=0.03). The authors concluded pretransplant assessment of donor specific and nonspecific immune parameters may identify recipients who can benefit from closer clinical and immunological surveillance to allow for tailored immunosuppression and selective intervention aimed at optimizing both short and long-term graft outcome. The use of the immune cell function assay as described in this study has not been cleared by the FDA.

Bhodrade and colleagues (2008), in an observational study, assessed the functional immune response in 143 sequential blood samples from 57 lung transplant recipients using the ImmuKnow assay. The assay was performed during a routine clinic visit when each subject was on a stable dose of tacrolimus. Study results demonstrated the average ImmuKnow assay in stable lung transplant recipients was 244 +/- 138 adenosine triphosphate (ATP) ng/ml and the median level was 236 ATP ng/ml. approximately 703 +/- 695 days after lung transplantation. There was no correlation found between ImmuKnow levels and tacrolimus dose or trough levels. Fifteen of the 57 lung transplant recipients developed infections and had a lower ImmuKnow level at the time of their infections as compared with stable lung transplant recipients (111 +/- 83 versus 283 +/- 143 ATP ng/ml, respectively, p = 0.0001). Sixteen of the remaining 42 recipients had low ImmuKnow assay values (< 225 ATP ng/ml), but did not have infections. Since the ImmuKnow assay levels were lower in infected lung transplant recipients compared with non-infected recipients and increased with treatment of these infections, it is unclear if the ImmuKnow assay reflected those who were over-immunosuppressed and at risk of infection or suppression of the bone marrow due to infectious agents. The authors concluded that further study of this assay is needed to determine its role optimizing immunosuppression in a given transplant recipient.

Cabrera and colleagues (2009), in a prospective single-center study, evaluated 42 consecutive liver transplant recipients for hepatitis C virus (HCV)-related end-stage liver disease. Blood samples were collected immediately prior to a liver biopsy being performed on each recipient and the blood was tested with the ImmuKnow assay. In those subjects whose liver biopsy showed signs of rejection, the immune response was noted to be high and in those with active recurrence of HCV, the immune response was low. The authors indicated that they did not see the Immuknow assay as a replacement for liver biopsy in the near future and recommended that further studies with larger populations of transplant recipients be performed to confirm their findings.

Husain and colleagues (2009) reported on the correlation between the ImmuKnow values and various infectious syndromes in a prospective cohort of lung transplant recipients. A total of 175 lung transplants with 129 infectious episodes were evaluated. The median ImmuKnow values in cytomegalovirus disease, viral infection, and bacterial pneumonia were found to be significantly different from stable state. The median ImmuKnow values of fungal disease and tracheobronchitis had a tendency to be lower than stable state. Of the transplant recipients colonized with fungus who subsequently developed fungal disease within 100 days, the median value of ImmuKnow was significantly lower than in those who did not develop fungal disease. The authors concluded that this is the first demonstration in a prospective cohort of lung transplant recipients in which lower ImmuKnow values independently correlate with increased risk of infection. Study results also suggested that this test may potentially be useful in identifying those with fungal colonization. Further testing is needed in order to validate results.

Kobashigawa and colleagues (2010) studied the effect of the ImmuKnow assay when it was used on heart transplant recipients between two weeks and 10 years post transplant. A total of 864 assays from 296 recipients were analyzed. All transplant recipients were being treated with triple drug immunosuppression therapy. There were 38 episodes of infection and eight episodes of rejection noted during this study. The average assay score was found to be lower during infection than at a steady state of non infection. The average assay score was not significantly different during rejection than at a steady state of non rejection. The authors concluded that assay scores may be able to predict risk of infection in a heart transplant recipient, but the association between higher scores and rejection is inconclusive due to the small number of rejection episodes evaluated. The authors further noted that a study with a larger sample size of rejection episodes and a randomized trial in which immunosuppression could be prospectively guided by the assay are needed.

Xue and colleagues (2010) studied the immunity levels as measured by the ImmuKnow assay in Chinese liver transplantation recipients and its potential application in monitoring the risk of post-transplant infection. A total of 45 healthy Chinese and 106 adult liver transplant (LT) recipients were evaluated. LT recipients were grouped into stable status or infection according to their clinical diagnosis. Whole blood samples were collected and the CD4+ T cells were selected and their ATP value was subsequently assayed. The average ImmuKnow assay in infected LT recipients was 128 ± 84 ng/mL, significantly lower (P<0.05) than that in stable LTs (305 ± 149 ng/mL) or in normal adults (301 ± 101 ng/mL). The ImmuKnow values in LTs had a good negative correlation to infection clinically (r = -0.6217, P<0.001). Infectious risk was high when the ImmuKnow value was less than 130 ng/mL (odds ratio=13, 95% confidence interval 6.0-29.4, P<0.01). The sensitivity of low ImmuKnow values in post-transplant infection was 85.2% and specificity was 76.3%. The authors concluded that further investigation will determine the role of the ImmuKnow assay in tailoring immunosuppression and preemptive measures such as starting antifungal agents.

Huskey and colleagues (2010) noted that since the FDA review of the ImmuKnow assay, "few studies have clarified the value of the ImmuKnow assay in the clinical monitoring of patients after kidney transplant." Because many of these previous studies had small numbers of participants, the authors performed a large retrospective analysis of ImmuKnow results obtained over a five year period and compared those values with subsequent events of opportunistic infections (OI) and acute rejection (AR). A total of 1330 ImmuKnow assay values in 583 renal transplant recipients from a single institution from 2004 to 2009 were evaluated. The assay values of the transplant recipients were compared to that of a control population matched for time, post-transplantation, gender and age. In participants with OI (n=94) there were no differences in prior assay values compared to that of matched controls (386 versus 417 ng/ml, P = 0.24). In 47 participants with AR, there were also no differences detected in prior assay results (390 versus 432 ng/ml, P = 0.25) when compared with controls. "Low" values (less than or equal to 225 ng/ml) lacked sensitivity and specificity as a predictive test for subsequent OI, as did "strong" (greater than or equal to 525 ng/ml) values as a predictive test for subsequent AR. The authors concluded "ImmuKnow assay measures at a single time point did not identify individuals at risk for the future development of clinically significant events." They also noted that further studies are required to clarify the role of this test in immune monitoring of kidney transplant recipients.

The American Society of Transplantation (AST) (2006) does not include the use of the Cylex Immune Cell function assay in its publication: "Recommendations for Screening, Monitoring and Reporting of Infectious Complications in Immunosuppression Trials in Recipients of Organ Transplantation."

Other Proposed Uses (not included in the FDA-label)

Augustine and colleagues (2007) reported that individuals with T-cell immunodeficiency diseases, including severe combined immunodeficiency disease (SCID) may have defective lymphocyte responses. They also noted that individuals with malignancies following bone marrow transplantation, with severe viral infections, or undergoing immunosuppressive therapy for others reason, may have severe suppression of cell mediated responses. The traditional "gold standard" method for testing cell-mediated immune function is the lymphocyte proliferation assay with incubation times varying from four to six days. The authors compared the lymphocyte proliferation assay with the immune cell function assay known as the ImmuKnow assay which has an overnight incubation period. Whole blood from 20 individuals suspected of having cell-mediated immunity defects and 21 normal controls were included in this study. Exact diagnoses of the individuals were not known because of "Health Insurance Portability and Accountability Act (HIPPA) regulations" but it was known that one individual had SCID. Of the 20 samples tested from individuals with suspected cell-mediated immunity defects, nine (45%) exhibited normal responses in both assays and seven samples (35%) had results considered as very low in both assays. There was agreement between both assays in 16 out of 20 (80%) of the individuals. Whole blood samples taken from the 21 normal controls were also tested using the ImmuKnow assay and the lymphocyte proliferation assay. Twenty (95%) of the 21 controls had results that agreed in both assays. Nineteen (90%) of the controls had both normal proliferation and moderate to strong responses. Results from one individual with SCID demonstrated very low responses in both methods. The authors noted that the ImmuKnow assay may be a useful screening tool for more rapid detection of blood samples with decreased cell-mediated immune responses. However, they did not propose that it be utilized to replace the traditional method but to serve as a rapid 18-24 hour screen prior to the traditional five to seven day proliferation assay. Study limitations include a small sample size.

Gesundheit and colleagues (2010) studied immune monitoring post allogeneic hematopoietic stem cell transplant (alloHSCT). The authors noted "after alloHSCT, immunosuppressed patients are susceptible to opportunistic infections, and uncontrolled function of the graft can result in graft versus host disease." Between October 2005 and November 2007, a total of 170 blood samples were collected from 40 individuals post alloHSCT performed for various malignant (31) and nonmalignant (9) diseases and from 13 healthy controls. The ImmuKnow assay was utilized for CD4 ATP levels to compare known clinically immunocompromised versus immunocompetent participants after alloHSCT. The researchers also compared the reconstitution of white blood cell count to the ImmuKnow results and clinical status. The participants' clinical course correlated with the stratification of immune response established by the ImmuKnow assay for solid organ transplantation (immunocompetent versus immunocompromised), and this sometimes differed from their white blood cell count. The authors concluded that the ImmuKnow assay should be evaluated prospectively in clinical trials.

Manga and colleagues (2010) reported on a study of 16 adults with hematologic malignancies undergoing hematopoietic stem cell (HSC) mobilization prior to autologous transplantation in which the immune cell function assay was used to measure ATP activity. Mobilization of HSC was achieved using hematopoietic growth factors (such as granulocyte colony stimulating factors [G-CSF]). The immune cell function assay measured the ATP activity in G-CSF treated individuals. The ATP activity was found to be significantly higher than that measured in healthy or "nonmobilized" individuals. The authors concluded that "the significance of ATP production by CD4⁺ cells in individuals with hematologic malignancies should be investigated in larger studies."

Clinicaltrials.gov lists two nonsolid organ transplant related studies assessing the ImmuKnow assay. One study was performed on allogeneic hematopoietic stem-cell transplant recipients and the other an industry sponsored trial to measure ATP expression in lymphocytes of individuals with multiple sclerosis undergoing various therapies by means of using the "ImmuKnow" test. The first study was terminated at the request of the sponsor and the second has been completed without study results published to date. ImmuKnow has also been studied in a number of other conditions including post-transplant lymphoproliferative disease (PTLD), rheumatoid arthritis, inflammatory bowel disease, and human immunodeficiency virus (HIV).

Conclusion

Currently, evidence is insufficient to conclude that individualized titration of immunosuppressive therapy based on the results of immune cell function testing results in improved clinical outcome in individuals following solid organ transplant. Furthermore, the utility of the assay to identify individuals at risk for rejection prior to kidney or any other solid organ transplant or for the management of other conditions is currently unproven. Further studies with larger populations are needed.

According to the United Network for Organ Sharing (2011), there were 28,664 solid organ transplants performed in the United States in 2010. Immunosuppressive drugs are needed to prevent and treat organ transplant rejection. Without immunosuppressive drugs, organ transplantation would almost always cause an adverse immune response and organ rejection.

The immune cell function assay is proposed to assess the immune function of the transplant recipient in order to individualize therapy. It has also been investigated as a method of identifying those at risk for early acute kidney transplant rejection prior to the actual kidney transplant and for the evaluation of a variety of other conditions such as autologous and allogeneic hematopoietic stem-cell transplant recipients, PTLD, immunodeficiency diseases including SCID and HIV, and autoimmune diseases including multiple sclerosis and inflammatory bowel disease.

A biopsy of the transplanted organ can confirm rejection. A routine biopsy is also often performed to detect rejection early, before symptoms develop. When organ rejection is suspected additional tests may be performed prior to organ biopsy. The testing is dependent on the type of transplant and may include:

• Abdominal CT scan
• Bronchoscopy
• Chest x-ray
• Heart echocardiography
• Kidney arteriography
• Kidney ultrasound
• Lab tests of kidney or liver function

The Cylex ImmuKnow assay received FDA clearance through the 510(k) process in 2002 for the detection of CMI response in populations undergoing immunosuppressive therapy for organ transplant. Evidence is insufficient at this time to establish whether or not the immune cell function assay is as beneficial as the established alternatives or results in improved clinical outcomes for any indication.

Allogeneic stem cells: Stem cells harvested from a donor.

Autologous stem cells: Stem cells harvested from the individual's own bone marrow prior to the cytotoxic therapy.

Cell mediated immunity: An immune response caused by killer cells, not antibodies.

Cytotoxic: Destructive to cells.

Graft versus host disease: A life-threatening complication of bone marrow transplant in which the donated marrow causes an immune reaction against the recipient's body.

Phytohemagglutinin (PHA): Lectins (extract of kidney beans) capable of causing erythrocytes and leukocytes to clump together; a global stimulator of the immune system.

The following codes for treatments and procedures applicable to this document are included below for informational purposes.  Inclusion or exclusion of a procedure, diagnosis or device code(s) does not constitute or imply member coverage or provider reimbursement policy.  Please refer to the member's contract benefits in effect at the time of service to determine coverage or non-coverage of these services as it applies to an individual member. 

When services are Investigational and Not Medically Necessary:
When the code describes a procedure indicated in the Position Statement section as investigational and not medically necessary.

Peer Reviewed Publications:

1. Augustine NH, Pasi BM, Hill HR. Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutinin. J Clin Lab Anal. 2007; 21(5):265-270.
2. Bhorade SM, Janata K, Vigneswaran WT, et al. Cylex ImmuKnow assay levels are lower in lung transplant recipients with infection. J Heart Lung Transplant. 2008; 27(9):990-994.
3. Cabrera R, Ararat M, Soldevila-Pico C, et al. Using an immune functional assay to differentiate acute cellular rejection from recurrent hepatitis C in liver transplant patients. Liver Transpl. 2009; 15(2):216-222.
4. Cadillo-Chávez R, de Echegaray S, Santiago-Delpín EA, et al. Assessing the risk of infection and rejection in Hispanic renal transplant recipients by means of an adenosine triphosphate release assay. Transplant Proc. 2006; 38(3):918-920.
5. Elidemir O, Kancherla BS, Schecter MG, et al. Post-transplant lymphoproliferative disease in pediatric lung transplant recipients: recent advances in monitoring. Pediatr Transplant. 2009; 13(5):606-610.
6. Gesundheit B, Budowski E, Israeli M, et al. Assessment of CD4 T-lymphocyte reactivity by the Cylex ImmuKnow assay in patients following allogeneic hematopoietic SCT. Bone Marrow Transplant. 2010; 45(3):527-533.
7. Husain S, Raza K, Pilewski JM, et al. Experience with immune monitoring in lung transplant recipients: correlation of low immune function with infection. Transplantation. 2009; 87(12):1852-1857.
8. Huskey J, Gralla J, Wiseman AC. Single Time Point Immune Function Assay (ImmuKnow^TM) Testing Does Not Aid in the Prediction of Future Opportunistic Infections or Acute Rejection. Clin J Am Soc Nephrol. 2010 Nov 18. [Epub ahead of print]
9. Kobashigawa JA, Kiyosaki KK, Patel JK, et al. Benefit of immune monitoring in heart transplant patients using ATP production in activated lymphocytes. J Heart Lung Transplant. 2010; 29(5):504-508.
10. Kowalski RJ, Post DR, Mannon RB, et al. Assessing relative risks of infection and rejection: a meta-analysis using an immune function assay. Transplantation. 2006; 82(5):663-668.
11. Manga K, Serban G, Schwartz J, et al. Increased adenosine triphosphate production by peripheral blood CD4⁺ cells in patients with hematologic malignancies treated with stem cell mobilization agents. Hum Immunol. 2010; 71(7):652-658.
12. Reinsmoen NL, Cornett KM, Kloehn R, et al. Pretransplant donor-specific and non-specific immune parameters associated with early acute rejection. Transplantation. 2008; 85(3):462-470.
13. Xue F, Zhang J, Han L, et al. Immune cell functional assay in monitoring of adult liver transplantation recipients with infection. Transplantation. 2010; 89(5):620-626.

Government Agency, Medical Society, and Other Authoritative Publications: 

1. Biogen Idec. ATP Expression in Lymphocytes of MS Patients by Means of "ImmuKnow^®" Assay. (Cylex). NLM Identifier: NCT00618267. Last updated on March 4, 2010. Available at: http://clinicaltrials.gov/ct2/show/NCT00618267?term=immuKnow&rank=2. Accessed on April 13, 2011.
2. Humar A, Michaels M; AST ID Working Group on Infectious Disease Monitoring. American Society of Transplantation recommendations for screening, monitoring and reporting of infectious complications in immunosuppression trials in recipients of organ transplantation. Am J Transplant. 2006; 6(2):262-274.
3. Indiana University School of Medicine. Investigation of the Cylex^® ImmuKnow^® Assay. NLM Identifier: NCT00569842. Last updated on: February 11, 2010. Available at: http://clinicaltrials.gov/ct2/show/NCT00569842?term=immuKnow&rank=1. Accessed on April 13, 2011.
4. U.S. Food and Drug Administration 510(k) Premarket Notification Database. Immune Cell Function Assay, Cylex Inc. Summary of Safety and Effectiveness. No. K101911. Rockville, MD. FDA. October 18, 2010. Available at: http://www.accessdata.fda.gov/cdrh_docs/pdf10/K101911.pdf. Accessed on April 6, 2011.

1. National Library of Medicine. MedLine Plus. Transplant rejection. Last update April 16, 2009. Available at: http://www.nlm.nih.gov/medlineplus/ency/article/000815.htm. Accessed on April 6, 2011.
2. United Network for Organ Sharing. Transplant data. Last updated March 5, 2010. Available at: http://www.unos.org/. Accessed on April 6, 2011.

Immune Cell Function Assay
ImmuKnow Assay

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