0773 Transplant Immune Cell Function Assays (1)

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Transplant Immune Cell Function Assays

Policy History

Last Review

10/27/2019

Effective: 01/09/2009

Next

Review: 08/27/2020

Review History

Definitions

Additional Information

Clinical Policy Bulletin

Notes

Number: 0773

Policy *Please see amendment for Pennsylvania Medicaid at the end of this CPB.

Aetna considers the ImmuKnow Assay, also known as the

Transplantation Immune Cell Function Assay (Cylex, Inc.,

Columbia, MD) medically necessary to adjudicate over

immunosuppression in subpopulations of transplant recipients

with co-morbid infection or cancer.

Aetna considers the ImmuKnow Assay experimental and

investigational for all other indications including any of the

following because of insufficient evidence of its effectiveness:

▪ For detection of cellular immune function in individuals

with renal cell carcinoma

▪ For identification of individuals at risk for rejection prior

to kidney, liver, lung, or any other solid organ transplant

▪ For management of individuals undergoing allogeneic

hematopoietic stem cell transplantation

▪ For management of individuals with inflammatory

bowel diseases (Crohn's disease and ulcerative colitis)

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▪ For management of organ transplant rejection in

individuals undergoing immunosuppressive therapy

post solid organ transplant

▪ For monitoring the immune response following surgery

▪ For prediction of infection risk in individuals receiving

disease-modifying anti-rheumatic drugs

▪ For prediction of risk infection in individuals with lupus

nephritis.

Aetna considers the Pleximmune test experimental and

investigational for prediction of acute cellular rejection in

children with liver or intestine transplantation and all other

indications because its clinical value has not been established.

Aetna considers the T-SPOT.CMV test experimental and

investigational because its clinical value has not been

established.

Aetna considers gamma interferon response experimental and

investigational for measurement of the bioactivity of

immunosuppressive medications in lung transplantation

because its clinical value has not been established.

Aetna considers measurement of donor-derived cell-free DNA

(e.g., Viracor TRAC) experimental and investigational as a

biomarker for allograft rejection in solid organ transplantation

because its clinical value has not been established.

Background

Transplant recipients have an increased risk of infection due to

the necessary immunosuppression. Conversely, under

immunosuppression carries the risk of rejection. Biopsy of the

transplanted organ can confirm rejection and is sometimes

performed before symptoms develop. When organ rejection is

suspected, additional tests may be performed prior to organ

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biopsy. Management of organ transplant rejection by an

immune cell function assay to assess the immune function of

the transplant recipients and to individualize therapy has been

proposed. It has also been investigated as a method of

identifying patients at risk for early acute kidney transplant

rejection prior to the actual kidney transplant.

In April 2002, the Food and Drug Administration (FDA) cleared

for marketing the Cylex Immune Cell Function Assay

(Cylex Inc., Columbia, MD) for the detection of cell mediated

immune response in populations undergoing

immunosuppressive therapy for organ transplant. According

to the 510(k) application submitted by the manufacturer to the

FDA, the test detects cell-mediated immunity in whole blood

after a 15 to 18 hour incubation with a stimulant (i.e.,

phytohemagglutinin). During incubation, increased adenosine

triphosphate (ATP) synthesis occurs within the cells that

respond to phytohemagglutinin. Concurrently, whole blood is

incubated in the absence of phytohemagglutinin for the

purpose of assessing basal ATP activity. Anti-CD4

monoclonal antibody coated magnetic particles are added to

immuno-select CD4 cells from both the stimulated and non-

stimulated cells. After washing the selected CD4 cells on a

magnet tray, a lysis reagent is added to release intracellular

ATP. Addition of luminescence reagent (luciferin/luciferase) to

the released ATP produces light measured by a luminometer,

which is proportional to the concentration of ATP. The

concentration of ATP (ng/ml) is calculated from a calibration

curve and compared to ATP level ranges to characterize the

cellular immune function of the sample.

Kowalski et al (2006) assessed the relative risks of infection

and rejection of 504 solid organ transplant recipients (heart,

kidney, kidney-pancreas, liver and small bowel) using the

ImmuKnow 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

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occurred. Odds ratios of infection or rejection were calculated

based on measured immune response values. The authors

reported that a recipient with an immune response value of 25

ng/ml ATP was 12 times (95 % confidence interval [CI]: 4 to

36) more likely to develop an infection than a recipient with a

stronger immune response and that a recipient with an

immune response of 700 ng/ml ATP was 30 times (95 % CI: 8

to 112) more likely to develop a cellular rejection than a

recipient with a lower immune response value.

Thai et al (2006) compared pancreas recipient clinical states

(stable, rejection, infection) with T cell responses using the

ImmuKnow assay. Blood samples were taken from pancreas

recipients pre-transplant and at approximately 3-month

intervals post-transplant for analysis of T cell responses.

When possible, T cell responses were also quantified during

changes in clinical status (infection or rejection). A range of

100 to 300 ng/ml ATP was found in stable patients (mean 194

+/- 123, n = 51) with good graft function and no infection or

rejection. A low T cell response correlated highly with

infectious states. Fourteen patients with infections/post

transplant lymphoproliferative disease had a mean ATP of 48

ng/ml. Risk hazard analysis showed that patients with ATP

levels less than 100 ng/ml were 4 to 7 times more susceptible

to infection compared to stable patients. Four patients with

rejection showed a T cell response of 550 ng/ml ATP, which

was statistically significant compared to stable patients,

although the sampling numbers (n = 9) were too small to be

conclusive.

Cadillo-Chavez and colleagues (2006) reviewed the records of

64 kidney transplant patients 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. Of the 58

patients that had pre-transplant and post-transplant ATP levels

tested, the authors reported no association between ATP

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levels and immunosuppression type, doses, or levels;

creatinine levels; white blood cell counts; HLA; and panel-

reactive antibody (p > 0.05). However, patients with moderate

or high pre-transplant ATP levels had more rejection episodes

(8/10) while patients with ATP levels in the low immune

response had more infections (6/11) (p < 0.001; relative risk

[RR] for rejection = 1.2; RR for infection = 4.4). 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 (ANOVA, p =

0.0145). Although acute rejections occurred mostly above

300, this was not significant (p = 0.059; RR = 0.9). Infections

were more frequent with ATP under 300 (RR = 7.3) and

severe infection (e.g., endocarditis, meningitis, peritoneal

abscesses, pneumonia, etc) were more frequent under 200 (p

< 0.001). When pre-transplant values were compared with

post-transplant values at the second week, an increase

correlated with rejection (p < 0.001, RR = 15.3), while a

decrease did not correlate with the infection (p = 0.845, RR =

1.4). Patients who received anti-rejection treatment had a

decrease in their ATP levels at day 5 (p = 0.002).

Batal et al (2008) reported on a retrospective study that found

a correlation between decreased immune cell function test

results and active BK virus replication, but not with acute

rejection, in kidney transplant recipients. ImmuKnow assay

measurements were performed on 15 samples from 8 patients

with BK viremia, 38 samples from 25 patients with BK viruria,

and 243 samples from 148 patients with no BK viruria or

viremia. The mean +/- SD amounts of ATP released in these

3 groups were 102.9 +/- 58.6, 227.2 +/- 146.4, and 231.8 +/

150.8 ng/ml, respectively (p = 0.002, viremia versus all other

samples). The investigators reported that, within the viruria

group, lower immune cell function assay values were

associated with higher urinary viral load (p = 0.037). There

was no significant relationship, however, between immune cell

function test results and acute transplant rejection. The

investigators concluded that prospective studies are needed to

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determine whether this assay can be used as a screening tool

to stratify patients by their ultimate risk of developing BK

viremia and BK virus nephropathy.

In a study of kidney transplant recipients, Serban et al (2009)

found that, although low ATP levels by ImmuKnow

assay identified patients at increased risk for infection, high

ATP values failed to correlate with rejection and did not justify

increased immunosuppression. The investigators assessed

the significance of immune cell function in 76 renal allograft

recipients after anti-thymocyte globulin induction and initiation

of maintenance immunosuppression. The investigators found

that the Immuknow assay yielded paradoxically high ATP

values during the first 3 months post-transplantation, despite

very low CD4+ counts. The investigators reported that high

ATP values were caused by peripheral blood myeloid cells, did

not predict rejection, and occurred primarily in transplant

recipients who received darbepoietin (p = 0.017). Over the

first 5 months post-transplantation, mean ATP activity

gradually decreased, whereas CD4+ counts slowly increased.

Low ATP values were predictive of infection (p = 0.002). The

investigators concluded that ImmuKnow results,

therefore need to be interpreted with caution in patients

receiving anti-thymocyte induction therapy; although low ATP

levels identified patients at increased risk for infection, high

ATP values failed to correlate with rejection and did not justify

increased immunosuppression.

A study by Bhorade et al (2008) found that the ImmuKnow

assay had high sensitivity but poor specificity for infection in

lung transplant recipients. The investigators identified the

level of functional immunity as measured by the ImmuKnow

assay in lung transplant recipients and correlated these values

with the dose and trough levels of immunosuppression as well

as other clinical parameters in these patients. The

investigators assessed the functional immune response in 143

sequential blood samples from 57 lung transplant recipients

using the ImmuKnow assay and reported that the average

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ImmuKnow assay in stable lung transplant recipients was 244

+/- 138 ATP ng/ml and the median level was 236 ATP ng/ml

(range 5 to 669 ATP ng/ml), about 703 +/- 695 days after lung

transplantation. There was no correlation between ImmuKnow

levels and tacrolimus trough levels. Stepwise multiple

regression analysis identified African American race as an

independent predictor of ImmuKnow assay levels when age,

gender and underlying diagnosis were taken into account (p <

0.04). The investigators found that the ImmuKnow assay

levels were lower in infected lung transplant recipients

compared with non-infected recipients and increased with

treatment of these infections. Fifteen infected lung transplant

recipients had a lower ImmuKnow level at the time of their

infections as compared with stable lung transplant recipients

(111 +/- 83 versus 283 +/- 143 ATP ng/ml, respectively, p =

0.0001). Sixteen of the remaining 42 patients had low

ImmuKnow assay values (less than 225 ATP ng/ml), but did

not have active infection. The investigators found that, while

the sensitivity for infection of an ATP value of less than 225

ng/ml was 93 % in this study, the specificity was only 38 %. In

addition, the utility of ATP measurements was not assessed,

as only 2 recipients in the patient sample had rejection. The

authors concluded, "It remains unclear whether the ImmuKnow

assay reflects over-immunosuppressed individuals at risk of

infection or bone marrow suppression by infectious agents.

Further investigation will determine the role of the ImmuKnow

assay in tailoring immunosuppression in lung transplant

recipients."

Husain et al (2009) reported the correlation between Cylex

ImmuKnow (ng/ml ATP) values and various infectious

syndromes in a large prospective cohort of lung transplant

recipients. These investigators followed-up 175 lung

transplants that developed 129 infectious episodes. Multiple

logistic regression analysis was performed; generalized

estimating equations were used to determine the odds ratio

(OR) for infections. The median ImmuKnow values in

cytomegalovirus disease (49.3 ng/ml ATP), viral infection (70

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ng/ml ATP), and bacterial pneumonia (92 ng/ml ATP) were

significantly different from stable state (174.8 ng/ml ATP). The

median ImmuKnow values of fungal disease (85 ng/ml ATP)

and tracheobronchitis (123 ng/ml ATP) had a tendency to be

lower than stable state (p = 0.10), whereas patients with fungal

colonization had comparable ImmuKnow values (167 versus

174.8 ng/ml ATP). Of the patients 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 (22.5 versus 183.5

ng/ml ATP; p < 0.0001). Generalized estimating equation

regression analysis showed ImmuKnow values less than or

equal to 100 ng/ml ATP to be an independent predictor of

infections (OR of 2.81). The authors concluded that Cylex

ImmuKnow assay monitoring has the potential to identify the

patients at risk of developing infection and those colonized

with fungus that are at risk of developing disease.

Another study, published in abstract form, demonstrated a very

poor correlation between histologically proven rejection and

the ImmuKnow assay, with 87 % of the allograft rejection

episodes occurring in the setting of a low to moderate ATP

level (Huang et al, 2007).

Cabrera et al (2009) used the ImmuKnow assay to help

assess the etiology of abnormal liver function test results in

liver transplant recipients. Blood samples for the immune

functional assay were taken from 42 recipients prospectively at

various times post-transplant and compared with clinical and

histologic findings. In patients whose liver biopsy showed

evidence of cellular rejection, the immune response was noted

to be very high, whereas in those with active recurrence of

hepatitis C, the immune response was found to be very low.

This finding was found to be statistically significant (p <

0.0001). In those patients in whom there was no predominant

histologic features suggesting 1 entity over the other, the

immune response was higher than in those with aggressive

hepatitis C but lower than in those with cellular rejection. The

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authors concluded that these data show the potential utility of

the ImmuKnow assay as a means of distinguishing hepatitis C

from cellular rejection and its potential usefulness as a marker

for outlining the progression of hepatitis C.

Macedo et al (2009) investigated the impact of Epstein-Barr

virus (EBV) load on T-cell immunity from pediatric

transplantation recipients, using clinically applicable tests for

improved assessment of T-cell immune competence. A total

of 35 asymptomatic pediatric thoracic transplantation patients

were categorized into 3 groups according to their EBV load

levels: (i) undetectable viral load (UVL), (ii) chronic low viral

load (LVL) and (iii) chronic high viral load (HVL). Global and

EBV-specific T-cell immunity were assessed by ATP release

using Cylex Immuknow and T Cell Memory assays. Patients

with UVL exhibited normal ATP release to Concanavalin A

(ConA) and phytohemagglutinin (PHA; 190 +/- 86 ng/ml, 328

+/- 163 ng/ml) and detectable EBV-specific (37 +/- 34 ng/ml)

ATP responses. Patients with LVL displayed significantly

stronger responses to ConA (373 +/- 174 ng/ml), PHA (498 +/

196 ng/ml) and EBV (152 +/- 179 ng/ml), when compared with

UVL or to HVL patients (ConA 185 +/- 114 ng/ml, PHA 318 +/

173 ng/ml, and EBV 33 +/- 42 ng/ml). Moreover, patients with

HVL displayed significant inverse correlation between CD4+

T-cell ATP levels and EBV loads. The authors concluded that

evaluation of global and EBV-specific T-cell immunity provides

a rapid assessment of patients' immune competence.

However, it is still unclear if selective over-suppressed ATP

release by CD4+ T cells reflects HVL patients at risk of post-

transplant lymphoproliferative disease. They stated that

further longitudinal studies will determine the importance of

Immuknow test in identifying asymptomatic HVL patients

vulnerable to EBV complications.

Rossano et al (2009) tested the hypothesis that the Cylex

ImmuKnow cell function assay (CICFA) is a clinically useful

test in pediatric heart transplant patients. All children

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undergoing heart transplantation at the study center (1989 to

2006) for whom CICFA levels were obtained were reviewed.

The association of CICFA levels with episodes of acute

rejection (AR) and significant infections was determined.

Among 83 patients (34 girls, 41 %), 367 CICFA levels were

obtained (median of 4.0; interquartile range [IQR], 2.0 to 6.0

per patient). There were 26 episodes of AR in 17 patients (20

%) and 38 infections in 34 patients (41 %). CICFA levels were

similar among patients with AR at the time of the CICFA

measurement (median of 325 [IQR, 163 to 480] ATP ng/ml)

versus patients without AR (median of 330 [IQR, 227 to 441]

ATP ng/ml; p = 0.36). CICFA levels were similar among

patients with infections within 1 month of CICFA measurement

(median of 295 [IQR, 216 to 366] ATP ng/ml) and those

without infections (median of 330 [IQR, 226 to 453] ATP ng/ml;

p = 0.24). The authors concluded that the CICFA is not

predictive of AR or significant infections in pediatric heart

transplant patients. On the basis of the available evidence,

these investigators stated that this assay can not be

recommended as part of the routine management of pediatric

heart transplant patients.

Gupta et al (2008) reported that the ImmuKnow assay had

limited clinical utility as an adjunct to routine clinical evaluation

in assessing risk of infection or rejection in heart transplant

recipients. The authors performed a retrospective review of the

clinical course of all adult cardiac transplant recipients who

underwent an ImmuKnow assay at University of Texas

Southwestern Medical Center between January 2004 and

September 2007. The authors reported that 111 patients were

free of significant rejection or infection at the time of the

first ImmuKnow assay. Most patients (92 %) were more than 1

year post-transplant. Over the next 157 +/- 41 (mean +/- SD)

days, 2 patients had 3 episodes of rejection requiring therapy

and 7 patients had 8 infections requiring therapy.

The ImmuKnow responses ranged from 17 to 894 ng/ml. No

correlation was observed between the baseline ImmuKnow

response and subsequent risk of either infection or rejection

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within 6 months. Lower white blood cell count and African

American ethnicity were correlated with a lower ImmuKnow

response. The authors concluded that the Cylex assay had

limited utility as an adjunct to routine clinical evaluation in

assessing risk of infection or rejection in cardiac transplant

recipients.

Gesundheit et al (2010) stated that following allogeneic

hematopoietic stem cell transplantation (alloHSCT),

immunosuppressed patients are susceptible to opportunistic

infections, and uncontrolled function of the graft can result in

graft-versus-host disease (GVHD). Accurate immune

monitoring may help early detection and treatment of these

severe complications. Between October 2005 and November

2007, a total of 170 blood samples were collected from 40

patients after alloHSCT in the Hadassah Hebrew University

Medical Center and from 13 healthy controls. These

researchers utilized the Cylex ImmuKnow assay for CD4 ATP

levels to compare known clinically immunocompromised

versus immunocompetent patients after alloHSCT. They also

compared the reconstitution of white blood cell (WBC) count to

the ImmuKnow results and clinical status. The patients'

clinical course correlated with the stratification of immune

response established by the ImmuKnow assay for solid organ

transplantation (immunocompetent versus

immunocompromised), and this often differed from their WBC

count. The authors concluded that the Cylex ImmuKnow

assay should be evaluated prospectively in clinical trials.

The American Society of Transplantation (AST) does not

mention the use of the Cylex Immune Cell Function assay in

its recommendations for the screening, monitoring and

reporting of infections complications in the evaluation of

recipients of organ transplantation (AST, 2006). Chon and

Brennan (2009) commented that there is no consensus on the

utility of the Immuknow assay in renal transplant

rejection other than in the research setting. Martinu et al

(2009) commented that "the data in lung transplantation are

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scarce and not very promising to date", and that "the

ImmuKnow assay does not seem to have the potential to

differentiate between infection and rejection in lung transplant

recipients and, until more data becomes available, should not

be used clinically in this patient population."

Bennett et al (2010) noted that infection of a transplanted

kidney with the polyomavirus, BK, is associated with poor

allograft survival. In an attempt to prevent this transplant

complication, these investigators studied 144 consecutive

transplant recipients for the presence of BK infection with

plasma and urine polymer chain reaction (PCR) testing at 1, 2,

3, 6 and 12 months. Viruria alone was followed by serial

studies. If plasma PCR became positive at greater than 2.6

log copies, mycophenolate was reduced until there was no

detectable plasma viral load. Urine PCR was positive in 34

(24 %), while plasma PCR turned positive in 22 cases (15 %).

No patients developed viremia with less than 6.8 log copies in

the urine. Viremia resolved within 3 months or less in 20 of 22

patients after reduction of immunosuppression. Surveillance

biopsies at 2 and 6 months revealed no BK nephropathy.

Eight patients had acute rejection during reduced

immunosuppression; however, all of these reversed with pulse

steroids. Patient and graft survival at 1 year was 99 % and 98

%, respectively. Use of the cell-mediated immunity assay

(ImmuKnow) was not useful in identifying infected patients.

Kobashigawa and colleagues (2010) examined the utility of

ImmuKnow in heart transplant recipients. Between November

2005 and July 2008, a total of 296 heart transplant recipients

had a total of 864 immun monitoring (IM) assays performed at

2 weeks to 10 years post-transplant and were correlated with

infection and rejection events that occurred within 1 month

after IM testing. All patients received standard triple-drug

immunosuppressive therapy with tacrolimus, mycophenolate

mofetil and corticosteroids, without induction therapy. There

were 38 infectious episodes and 8 rejection episodes. The

average IM score was significantly lower during infection than

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steady state (187 versus 280 ng ATP/ml, p < 0.001). The

average IM score was not significantly different during

rejection when compared with steady state (327 versus 280 ng

ATP/ml, p = 0.35). Interestingly, 3 of 8 rejection episodes

were antibody-mediated rejections and had hemodynamic

compromise and, for these, the mean IM score was

significantly higher than for steady-state patients (491 versus

280 ng ATP/ml, p < 0.001). The authors concluded that

ImmuKnow appears to predict infectious risk in heart

transplant patients. The association between high IM scores

and rejection risk is inconclusive due to the small number of

rejection episodes. They stated that further studies with larger

sample sizes for rejection episodes are needed.

Torio et al (2011) stated that the Cylex ImmuKnow assay

provides a rapid assessment of global immune function in

immunocompromised patients by measuring the global

immune responses of CD4 T cells from a whole-blood sample.

It may help to monitor the immune status of

immunosuppressed transplant patients. However, earlier

studies have shown that there is no consensus on the utility of

the ImmuKnow assay in renal transplant rejection. T-cell

activation was determined by measuring an increase of

intracellular ATP (iATP) from CD4 cells in 227 samples from

116 kidney transplant patients. The results were analyzed

regarding patient clinical status, namely, rejection, infection, or

stability. In addition, these researchers measured the

immunologic response of 108 healthy control subjects. There

were 24 infectious and 36 rejection episodes. Intracellular

ATP concentrations differed significantly between stable and

infected patients (180.5 +/- 55.2 versus 375.3 +/- 140.1 ng/ml;

p < 0.001) and between infected patients and control subjects

(180.5 +/- 55.2 versus 436.5 +/- 112 ng/ml; p < 0.001). No

correlation was observed between patients suffering an acute

rejection episode with this response. The authors concluded

that these findings confirmed that the ImmuKnow assay

identified transplant patients at risk for infection. It may

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provide information to guide immunosuppressive therapy, but

the assay did not seem to have the potential to differentiate

subjects experiencing rejection.

De Paolis et al (2011) evaluated the value of ImmuKnow (IK),

a new tool to measure the net state of immune function among

renal transplant recipients, in correlation with clinical and

laboratory data among unselected renal transplant recipients.

A total of 49 recipients of mean age of 51 years were enrolled

and followed for 1 year after transplantation. All subjects

received the same immunosuppressive strategy with

basiliximab induction and tacrolimus, mycophenolate mofetil

and steroid maintenance therapy. Samples for IK were

collected before transplantation as well as at 7, 14, 21 and 42

days and after 3, 6, and 12 months. There were 54 samples

with IK less than 225 ng/ml, 201 samples with normal IK

values, and 135 samples with greater than 525 ng/ml. These

investigators divided recipients into 3 groups with respect to

their basal IK values: Group 1 (Gr1; IK less than 225 ng/ml);

Group 2 (Gr2; normal values of IK between 226 and 524

ng/ml); and Group 3 (Gr3; IK greater than 525 ng/ml). At 1

year, these investigators observed a significant difference

among IK values at the start and the end of the study: Gr1

versus Gr2, p < 0.0001; Gr2 versus Gr3, p < 0.06 and Gr 1

versus Gr 3, p < 0.01). They observed reduced IK values to

predict an increased risk of infection, particularly with

cytomegalovirus (CMV) replication while higher IK value did

not correlate with an increased risk of acute rejection

episodes. Reduction of serum creatine levels occurred within

1 year in all groups (p < 0.005), but there was a significant

difference between Gr 2 versus Grs 1 and 3 (p < 0.0001 and p

< 0.0005, respectively). There findings suggested that more

stable IK values were associated with clinical quiescence and

laboratory stability. The authors concluded that this

preliminary analysis showed a beneficial capacity of this assay

to represent the global depression of the immune system.

They noted that reduced IK values, as a sign of excessive

immunosuppressive therapy, were associated with an

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increased risk of infection. They did not confirm the predictive

value of higher IK values for an increased risk of an acute

rejection episode.

Huskey et al (2011) retrospectively analyzed 1,330 ImmuKnow

assay values in 583 renal transplant recipients at a single

center from 2004 to 2009 and correlated these values with

episodes of opportunistic infections (OI) and acute rejection

(AR) in the subsequent 90 days. Assay values were

compared with a control population matched for age, gender,

and time post-transplantation. In patients with OI (n = 94),

there were no differences in prior mean assay values

compared with matched controls (386 versus 417 ng/ml, p =

0.24). In 47 patients with AR, again no differences were

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 that these findings fail

to show an association between single time point ImmuKnow

assay values and the subsequent development of an adverse

event in the subsequent 90 days. The optimal use of the

ImmuKnow assay in kidney transplantation has yet to be

determined.

Cheng et al (2011) determine the utility of the ImmuKnow

assay in assessing the risk of infection, rejection, and tumor

recurrence in liver transplant recipients. Immune function, as

determined by the ImmuKnow assay, was used to monitor the

global immune status in 342 whole blood samples from 105

liver transplant recipients. The association between ATP value

and post-transplant tumor recurrence was evaluated in 60

hepato-cellular carcinoma (HCC) patients. The ATP value in

predicting tumor recurrence in other independent cohort of 92

recipients with HCC was analyzed prospectively. The mean

ATP values of liver transplant recipients with infection

(145.2 +/- 87.0 ng/ml) or acute rejection (418.9 +/- 169.5

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ng/ml) were different from those with stable state (286.6 +/

143.9 ng/ml, p < 0.05). In recipients with HCC who developed

recurrent tumors, the values were significantly lower than

those without recurrence (137.8 +/- 66.4 versus 289 +/- 133.9

ng/ml, p < 0.01); the optimal threshold value to predict post-

transplant tumor recurrence was 175 ng/ml. Comparing with

the patients in lower immune group (ATP les than or equal

to 175 ng/ml), patients in the higher immune group

(ATP greater than 175 ng/ml) experienced significantly better

disease-free survival (p < 0.01). Multi-variate Cox regression

analysis showed the ATP value was an independent predictor

of HCC recurrence. The authors concluded that the

ImmuKnow assay has the potential to evaluate the risk of

infection and rejection in liver transplantation and to predict

post-transplant tumor recurrence in recipients with HCC.

Rodrigo and colleagues (2012) conducted a systematic

literature review to identify studies documenting the use of

ImmuKnow to monitor immune function in liver transplant

recipients until March 2012. Study quality was assessed by

using the Quality Assessment of Diagnostic Accuracy Studies

2 score. These investigators identified 5 studies to analyze

ImmuKnow performance in infection and 5 in acute rejection.

Pooled sensitivity, specificity, positive likelihood ratio,

diagnostic odds ratio and area under a summary receiver-

operating characteristic curve were 83.8 % (95 % CI: 78.5 % to

88.3 %), 75.3 % (95 % CI: 70.9 % to 79.4 %), 3.3 (95 % CI: 2.8

to 4.0), 14.6 (95 % CI: 9.6 to 22.3) and 0.824 +/- 0.034 for

infection and 65.6 % (95 % CI: 55.0 % to 75.1 %), 80.4 % (95

% CI: 76.4 % to 83.9 %), 3.4 (95 % CI: 2.4 to 4.7), 8.8 (95 %

CI: 3.1 to 24.8) and 0.835 +/- 0.060 for acute rejection.

Heterogeneity was low for infection and high for acute

rejection studies. The authors concluded that ImmuKnow test

is a valid tool to know the risk of further infection in adult liver

transplant recipients. Moreover, they stated that significant

heterogeneity across studies precludes concluding that

ImmuKnow identifies liver transplant patients at risk for

rejection.

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Shino et al (2012) hypothesized that the ImmuKnow assay can

be used to assess the immune function of lung transplant

recipients and identify those at risk of developing acute cellular

rejection and respiratory infection. Lung transplant recipients

at University of California Los Angeles between January 1,

2006 and December 31, 2009 received a bronchoscopy with

broncheo-alveolar lavage, transbronchial biopsy and

ImmuKnow values drawn at regular intervals as well as during

episodes of clinical deterioration. The recipient's clinical

condition at each time-point was classified as healthy, acute

cellular rejection, or respiratory infection. Mixed-effects

models were used to compare the ATP levels among these

groups, and odds ratios for rejection and infection were

calculated. The mean ATP level was 431 +/- 189 ng/ml for the

rejection group versus 377 +/- 187 ng/ml for the healthy group

(p = 0.10). A recipient with an ATP level greater than 525

ng/ml was 2.1 times more likely to have acute cellular rejection

(95 % CI: 1.1 to 3.8). Similarly, the mean ATP level was

323 +/- 169 ng/ml for the infection group versus 377 +/- 187

ng/ml for the healthy group (p = 0.03). A recipient with an ATP

level less than 225 ng/ml was 1.9 times more likely to have

respiratory infection (95 % CI: 1.1 to 3.3). However, the test

was associated with poor performance characteristics. It had

low sensitivity, specificity with an area under the receiver

operating characteristic curve of only 0.61 to diagnose

rejection and 0.59 to diagnose infection. The authors

concluded that the ImmuKnow assay appears to have some

ability to assess the overall immune function of lung transplant

recipients. However, this study does not support its use as a

reliable predictor of episodes of acute cellular rejection or

respiratory infection.

In a meta-analysis, Ling et al (2012) evaluated the

effectiveness of the Cylex ImmuKnow cell function assay

(CICFA) in identifying risks of infection and rejection post-

transplantation. After a careful review of eligible studies,

sensitivity, specificity, and other measures of the accuracy of

CICFA were pooled. Summary receiver operating

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characteristic curves were used to represent the overall test

performance. A total of 9 studies met the inclusion criteria.

The pooled estimates for CICFA in identification of infection

risk were poor, with a sensitivity of 0.58 (95 % CI: 0.52 to

0.64), a specificity of 0.69 (95 % CI: 0.66 to 0.70), a positive

likelihood ratio of 2.37 (95 % CI: 1.90 to 2.94), a negative

likelihood ratio of 0.39 (95 % CI: 0.16 to 0.70), and a

diagnostic odds ratio of 7.41 (95 % CI: 3.36 to 16.34). The

pooled estimates for CICFA in identifying risk of rejection were

also fairly poor with a sensitivity of 0.43 (95 % CI: 0.34 to

0.52), a specificity of 0.75 (95 % CI: 0.72 to 0.78), a positive

likelihood ratio of 1.30 (95 % CI: 0.74 to 2.28), a negative

likelihood ratio of 0.96 (95 % CI: 0.85 to 1.07), and a

diagnostic odds ratio of 1.19 (95 % CI: 0.65 to 2.20). The

authors concluded that current evidence suggests that CICFA

is not able to identify individuals at risk of infection or

rejection. They stated that additional studies are still needed

to clarify the usefulness of this test for identifying risks of

infection and rejection in transplant recipients.

Akimoto et al (2013) examined the ability of the ImmuKnow

(assay to predict the risk of infection in rheumatoid arthritis

(RA) patients receiving synthetic or biological disease-

modifying anti-rheumatic drugs (DMARDs). The amount of

ATP produced by CD4+ cells in response to

phytohemagglutinin was measured in whole blood from 117

RA patients without infection versus 17 RA patients with

infection, and compared with results in 75 healthy

controls. The mean ATP level was significantly lower in

patients with infection compared to both healthy controls (p <

0.0005) and patients without infection (p = 0.040). Also, the

mean ATP level in patients without infection was significantly

lower than that in healthy controls (p = 0.012). There was no

correlation between the ATP level and the Disease Activity

Score in 28 joints. The authors concluded that the ImmuKnow

assay results may be effective in identifying RA patients at

increased risk of infection, but the results showed no

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correlation with RA activity. They stated that larger studies

are needed to establish the clinical advantages of this assay in

RA treatment.

There is insufficient evidence of the effectiveness of the

ImmuKnow assay in the management of organ transplant

rejection in individuals undergoing immunosuppressive

therapy post solid organ transplant and for the identification of

individual risk for rejection prior to kidney or any other solid

organ transplant. Prospective clinical outcome studies are

needed to determine its role in the management of solid organ

or stem cell transplant recipients.

Lopez-Hoyos et al (2013) stated that ImmuKnow is an in-vitro

diagnosis method that uses patient samples of whole blood

polyclonally stimulated with phytohemagglutinin. It also

measures ATP production by CD4+ T cells. The test aims to

offer an objective and overall measurement of each

individual's cellular immune response. The assay was

designed with the idea of individually monitoring the

immunosuppression administered to transplant patients. At

the same time, it aims to help achieve a balance as a way of

avoiding immunosuppression excess and the associated

adverse effects (infections, cancer, etc.) or an

immunosuppression defect and the subsequent risk of allograft

rejection. The majority of studies that have evaluated its

clinical usefulness display great diversity in terms of patient

recruitment, the immunosuppressant treatment received, the

clinical variables analyzed and, above all, the time between

performing ImmuKnow and the evaluated clinical event. The

most consistent data showed that this assay on CD4+ T cell

functioning is useful for predicting the risk of infection in renal

transplant patients. However, its use as a rejection risk

indicator is unclear. Lastly, given the great variability of

immune response amongst individuals and that of existing

publications, it can be deduced that the isolated ImmuKnow

value does not have diagnostic capacity and only individual

serial monitoring could provide definitive assistance in clinical

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decision making and immunosuppressant treatment changes.

Moreover, the authors stated that other aspects of ImmuKnow

application in the clinical routine, such as assay cycles, require

randomized prospective studies for more comprehensive

information.

Israeli et al (2013) noted that currently there is no standardized

non-invasive diagnostic tool for the evaluation of

immunological complications such as GVHD and for managing

the cellular immune function of the transplant recipient. The

ImmuKnow assay for cellular immune function monitoring has

been incorporated successfully into the clinical follow-up

routine of solid organ transplant recipients. These researchers

examined the relevance and potential contribution of immune

monitoring using the assay in the setting of HCT. They found

that ImmuKnow-level measurement can distinguish between

states of immune function quiescence and between events of

acute GVHD. ImmuKnow levels were significantly higher in

patients going through GVHD than the levels measured for the

same patients during immunological stability. Moreover, they

demonstrated a patient case where longitudinal monitoring

using the ImmuKnow assay provided a trustworthy depiction of

the patient's cellular immune function post-HCT. The authors

concluded that they provided evidence for the potential

contribution of the ImmuKnow assay for longitudinal

individualized cellular immune function monitoring of patients

following HCT. Moreover, they stated that further studies are

needed to establish the optimal practice for utilizing the assay

for this purpose.

Brandhorst et al (2013) stated that Crohn's disease (CD) and

ulcerative colitis (UC) are inflammatory bowel diseases (IBDs),

which are characterized by dysfunctional regulation of the

immune system. A number of immune modifying drugs are

used to treat CD and UC. Therapy is adjusted largely on the

bases of subjective reports of disease activity and non-specific

laboratory tests. Identification of a single or combination of

immune markers of disease activity could be useful to select

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and monitor therapeutic responses. However, to-date no

reliable quantitative associations between IBD activity and

laboratory measures of immune function have been identified.

These investigators evaluated the usefulness of ImmuKnow as

a surrogate marker of IBD activity. Adult IBD patients with

either CD (n = 55, 27 males, mean, SD age = 38.5, 11.5 years)

or UC (n = 45, 24 males, mean, SD age = 41.7, 15.4 years)

were enrolled. Patients both in clinical remission and with

active disease provided responses to structured, validated

questionnaires (CDAI and HBI for CD patients and SCCAI for

UC patients) used to monitor IBD activity. Whole blood and

plasma samples were collected to quantify various markers of

disease status including routine cell counts and differentials

(CBCs), C-reactive protein (CRP), and albumin (Alb), as well

as CD4(+) immune response (ImmuKnow, n = 98). Results

were compared between all IBD patients as well as between

CD and UC subgroups. There was a good correlation

between the results of CDAI and HBI scores (r = 0.811, p <

0.01, Spearman-Rho), but HBI scores correlated slightly better

(r = 0.575, p < 0.001) than the CDAI's (r = 0.449, p = 0.001)

with CD patients' reported perception of their general

condition. Furthermore, CDAI and HBI scores categorized

12/55 versus 36/55 of CD patients respectively as having

active disease; SCCAI scores indicated that 25/45 of UC

patients had active disease. ImmuKnow results (in ng/ml of

ATP) were increased in 74/98 IBD subjects (greater than or

equal to 525 ng/ml, but were influenced by the use of systemic

corticosteroids (SCS) and infliximab. There were weak but

statistically significant Spearman-Rho correlations between Alb

concentrations and both CDAI (r = 0.413, p = 0.002) and HBI

(r = 0.325, p = 0.017) scores as well as between CRP values

and HBI scores (r = 0.331, p = 0.016). Correlations between

CRP and both CDAI and SCCAI scores and between Alb and

SCCAI scores were not significant and there were no

significant positive associations between any of the 3 clinical

scores and ImmuKnow results. The authors concluded that

CD4(+) immune responses (ImmuKnow results) were

significantly elevated in IBD patients whether or not they were

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in clinical remission, but were influenced by treatment. There

were some significant correlations between the clinical scores

and CRP or Alb, but not with the CD4(+) results. Both other

clinical scoring systems, other measures of immune function,

and CD4(+) immune response changes over time should be

examined to see if this or other laboratory measures of

immune response are predictive of actual disease activity or

symptoms in CD or UC patients.

Wong et al (2014) stated that the ImmuKnow ICFA reports ex-

vivo CD4 lymphocyte activation to quantify

immunosuppression. Limited organ and age-specific data

exist for pediatric heart transplant recipients. These

investigators examined their normative values and ICFA's

association with rejection/infection. A total of 380 ICFAs from

58 heart transplant recipients (6.5/recipient) were studied

retrospectively. The median age at the time of their first ICFA

was 5.3 yrs (IQR 2.4 to 12.1 yrs). ICFA levels during

immunologic stability (n = 311) were a median of 305 (IQR:

172 to 483) and mean of 353 (S.D. ± 224) ng ATP/ml. ICFA

levels trended lower with advancing age. ICFA levels during

immunologic stability increased over time from transplant after

the first 6 months but were not correlated with calcineurin

inhibitor levels or the type used. There was no association

between ICFA values during stability and rejection (median of

368 ATP ng/ml; IQR 153 to 527) or infection (median of 293

ATP ng/ml; IQR 198 to 432). In contrast to the manufacturer's

suggested ranges, the immunologic stable ranges in pediatric

cardiac recipients were very different. The authors concluded

that ICFA values during immunologic stability are related to

time from transplant in pediatric heart recipients; ICFA's ability

to discriminate rejection or infection from immunologic stability

was not demonstrated.

Ryan et al (2014) noted that management of pediatric renal

transplant patients involves multi-factorial monitoring

modalities to ensure allograft survival and prevent

opportunistic infection secondary to immunosuppression. An

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ICFA, which utilizes CD4 T-cell production of ATP to assess

immune system status, has been used to monitor transplant

recipients and predict susceptibility of patients to rejection or

infection. However, the validity of this assay to reflect immune

status remains unanswered. In a 2-yr retrospective study that

included 31 pediatric renal transplant recipients, 42 patient

blood samples were analyzed for immune cell function levels,

creatinine, WBC (white blood cell) count, immunosuppressive

drug levels, and viremia, concurrent with renal biopsy. T-cell

ATP production as assessed by ICFA levels did not correlate

with allograft rejection or with the presence or absence of

viremia. ICFA levels did not correlate with serum creatinine or

immunosuppressive drug levels, but did correlate with WBC

count. The authors concluded that ICFA is unreliable in its

ability to reflect immune system status in pediatric renal

transplantation; further investigation is needed to develop

methods that will accurately predict susceptibility of pediatric

renal transplant recipients to allograft rejection and infection.

An UpToDate review on “Investigational methods in the

diagnosis of acute renal allograft rejection” (Chon and

Brennan, 2104) states that “The ImmuKnow assay is a US

Food and Drug Administration (FDA)-approved test intended

to estimate the net state of immune system in

immunocompromised patients. It measures the ability of CD4

cells to respond to mitogenic stimulation by

phytohemagglutinin-L in-vitro by quantifying the amount of

adenosine triphosphate (ATP) produced and released from

these cells following stimulation. At the present time, there is

no consensus on the utility of these tests, other than in the

research setting”.

Liu and colleagues (2014) stated that it is uncertain whether

ImmuKnow can predict the risk of infection in lupus nephritis

(LN) patients receiving immunosuppressive therapy. The

ImmuKnow Immune Cell Function Assay was applied to

measure the activity of CD4+ T cells, as a marker of global

immune-competence. The correlation between changes in T

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cell activation and the relative risk of over-immunosuppression

as well as infection was studied. The amount of ATP

produced by CD4+ T cells in response to PHA was measured

for 74 LN patients without infection, 22 LN patients with severe

infection (i.e., required hospitalization), and 28 healthy

controls. No correlation was found between the ATP level and

systemic lupus erythematosus (SLE) activity. The mean ATP

level was significantly lower in LN patients with infection than

that in healthy controls (p < 0.01) and non-infected LN patients

(p < 0.01). The mean ATP level in non-infected LN patients

was not significantly different compared to healthy controls. A

cut-off ATP value of 300 ng/ml predicted infection in LN

patients with a specificity of 77 % and a sensitivity of 77 %.

Multi-variable partial correlation coefficient between the ATP

assay and severe infection was r = -0.040, p < 0.001; CRP

was r = 0.962, p < 0.001. The authors concluded that the

ImmuKnow assay may be effective in identifying an increased

risk of infection in LN patients but is not correlated with SLE

activity. Combined CRP value will increase the diagnostic rate

of severe infection in SLE. Moreover, they stated that larger

studies are needed to establish clinical advantages of this

assay in SLE treatment.

Ravaioli and colleagues (2015) observed that an immune

function assay shows promise for identifying solid organ

recipients at risk for infection or rejection. These investigators

conducted a randomized prospective study to assess the

clinical benefits of adjusting immunosuppressive therapy in

liver recipients based on ImmuKnow immune function assay

results. Adult liver recipients were randomized to standard

practice (control group; n = 102) or serial immune function

testing (interventional group; n = 100) performed with the

ImmuKnow assay before transplantation, immediately after

surgery and at day 1, weeks 1 to 4, 6, and 8, and months 3 to

6, 9, and 12. The assay was repeated within 7 days of

suspected/confirmed rejection/infection and within 1 week after

event resolution. Based on immune function values,

tacrolimus doses were reduced 25 % when values were less

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than 130 ng/ml ATP (low immune cell response) and

increased 25 % when values were greater than 450 ng/ml ATP

(strong immune cell response). The 1-year patient survival

was significantly higher in the interventional arm (95 % versus

82 %; p < 0.01) and the incidence of infections longer than 14

days after transplantation was significantly lower among

patients in the interventional arm (42.0 % versus 54.9 %, p <

0.05). The difference in infection rates was because of lower

bacterial (32 % versus 46 %; p < 0.05) and fungal infection (2

% versus 11 %; p < 0.05). Among recipients without adverse

events, the study group had lower tacrolimus dosages and

blood levels. The authors concluded that ImmuKnow immune

function testing provided additional data which helped optimize

immunosuppression and improve patient outcomes.

Detection of Cellular Immune Function in Patients with Renal Cell Carcinoma

Zheng and colleagues (2015) examined the clinical value of

the CD4(+) T cell ATP levels in patients with renal cell

carcinoma through the application of the ImmuKnow assay.

These researchers recruited 104 patients with renal cancer

who had undergone surgery from March 2009 to June 2012,

and were subsequently treated by dendritic cell and cytokine

induced killer cell bio-therapy or interferon-alpha therapy. The

changes in CD4(+) T cell ATP levels were detected at the peri

operative period and at 10 days, 1 month, 3 months, and 1

year after the surgery using the ImmuKnow assay. In addition,

the differences in ATP levels in different therapy groups were

compared and the prognosis conditions were analyzed. The

results demonstrated that no significant difference in the ATP

levels occurred at different time-points. Furthermore, there

were no obviously different ATP levels between the different

therapy groups, and the ATP levels were found to have no

clinical significance for the assessment of renal cancer

prognosis. The authors concluded that the findings of this

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study suggested that CD4(+) T cell ATP levels as detected by

the ImmuKnow assay have no obvious clinical value in

patients with renal cancer.

Monitoring the Immune Response Following Surgery

Maki and associates (2015) measured ATP levels in CD4+ T

cells as a marker of T-cell activity following surgery for

colorectal cancer using the ImmuKnow assay kit. A total of 16

consecutive patients who underwent surgical resection for

colorectal cancer between August and December, 2012 were

enrolled in this study, of whom 7 underwent laparoscopic

resection and 9 underwent open abdominal surgery. The

intracellular ATP levels in CD4+ T-lymphocytes were

measured using the ImmuKnow assay kit pre-operatively and

on the 1st, 4th and 8th post-operative days, as were the WBC

count, lymphocyte count and CRP levels. The ATP level of the

CD4+ T-cells was significantly elevated on the 1st day

following surgery compared to the pre-operative level (p <

0.01) and gradually returned to pre-operative levels; the

lymphocyte count was significantly decreased on the 1st post

operative day (p < 0.001). In addition, the ImmuKnow assay

demonstrated that only the ATP level, but not the WBC count,

lymphocyte count or CRP level, exhibited a significant

difference on the 1st (p = 0.080) and 8th (p = 0.042) post

operative days between the laparoscopic and open abdominal

surgery groups. The authors concluded that the ATP level of

CD4+ T-lymphocytes was increased in response to surgical

stress, in tandem with a decrease in the lymphocyte count.

The authors concluded that the ImmuKnow assay kit may be

clinically applicable for monitoring the immune response

following surgery, as it exhibited a higher sensitivity compared

to other assays. These findings need to be validated by well-

designed studies.

The Pleximmune Test

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According to Plexision (Pittsburgh, PA), the Pleximmune test is

the only FDA-approved blood test that predicts rejection in

children with liver or intestine transplantation. The test

measures the risk of rejection by measuring the recipient's

immune response to the donor. The results are reported as an

individualized index; the sensitivity and specificity of this test

approaches or exceeds 80 %.

Sindhi and colleagues (2016) stated that the Pleximmune test

is the first cell-based test approved by the FDA, which predicts

acute cellular rejection in children with liver- or intestine

transplantation. The test addresses an unmet need to improve

management of immunosuppression, which incurs greater

risks of opportunistic infections and EBV-induced malignancy

during childhood. High-dose immunosuppression and

recurrent rejection after intestine transplantation also result in

a 5-year graft loss rate of up to 50 %. Such outcomes seem

increasingly unacceptable because children can experience

rejection-free survival with reduced immunosuppression. The

sensitivity and specificity of the Pleximmune test for predicting

acute cellular rejection are 84 % and 80 %, respectively in

training set-validation set testing of 214 children. Among

existing gold standards, the biopsy detects but cannot predict

rejection. Anti-donor antibodies, which presage antibody-

mediated injury, reflect late-stage allo-sensitization as a down

stream effect of engagement between recipient and donor

cells. Therefore, durable graft and patient outcomes also

require accurate management of cellular immune responses in

clinical practice.

An expert commentary of this review stated that “Alloantigen

specific CD154+T-cytotoxic memory cells (CD154+TcM)

measured in the Pleximmune blood test provide a

personalized measure of donor-specific cellular alloreactivity, a

universal mechanism of acute cellular rejection. Therefore, the

test system can potentially serve as a surrogate for this event,

and provide non-invasive detection or prediction of this event

in other organ systems … The potential of allospecific

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CD154+TcM to detect acute cellular rejection in other organ

systems is illustrated by evaluation of 43 adult renal transplant

recipients. All subjects were sampled at the time of ‘for cause’

biopsies for allograft dysfunction. The IR of CD154+TcM,

which was associated with ongoing ACR in 32 of 43 training

set subjects demonstrated a sensitivity and specificity of 88 %

each. In the remaining subjects test sensitivity and specificity

of 100 % and 88 % replicated test performance confirming

additional uses of allospecific CD154+TcM. This experience

suggests that it will be possible to assess rejection-risk in adult

recipients using the principles of the test system described

here”.

Ashokkumar and associates (2017) noted that allospecific

CD154+TcM predicted acute cellular rejection after liver

transplantation (LTx) or intestine transplantation (ITx) in small

cohorts of children and can enhance immunosuppression

management, but await validation and clinical implementation.

To establish safety and probable benefit, these investigators

measured CD154+TcM in cryopreserved samples from 214

children younger than 21 years (National Clinical Trial

1163578). Training set samples (n = 158) were tested with

research-grade reagents and 122 independent validation set

samples were tested with current good manufacturing

practices-manufactured reagents after assay standardization

and reproducibility testing. Recipient CD154+TcM induced by

stimulation with donor cells were expressed as a fraction of

those induced by HLA non-identical cells in parallel cultures.

The resulting immunoreactivity index (IR) if greater than 1

implies increased rejection-risk. Training and validation set

subjects were demographically similar. Mean coefficient of

test variation was less than 10 % under several conditions.

Logistic regression incorporating several confounding

variables identified separate pre-transplant and post-transplant

IR thresholds for prediction of rejection in the respective

training set samples. An IR of 1.1 or greater in post-transplant

training samples and IR of 1.23 or greater in pre-transplant

training samples predicted LTx or ITx rejection in

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corresponding validation set samples in the 60-day post-

sampling period with sensitivity, specificity, positive predictive

value (PPV), and negative predictive value (NPV) of 84 %, 80

%, 64 %, and 92 %, respectively (area under the receiver

operator characteristic curve, 0.792), and 57 %, 89 %, 78 %,

and 74 %, respectively (area under the receiver operator

characteristic curve, 0.848). No adverse events (AEs) were

encountered due to phlebotomy. The authors concluded that

allospecific CD154+T-cytotoxic memory cells predicted acute

cellular rejection after LTx or ITx in children; adjunctive use

can enhance clinical outcomes.

While the Pleximmune test results correlated with rejection,

they were based upon a small number of subjects in the FDA

labeling for the humanitarian device exemption (HDE) and a

published study, in which there were wide confidence intervals

for sensitivity, specificity as well as PPV and NPV.

Furthermore, there are currently no clinical outcome studies

(clinical utility) and no evidence-based guidelines supporting

the use of the Pleximmune test.

T-SPOT.CMV Test

According to Oxford Immunotec Inc., the T-SPOT.CMV test

measures the strength of T cell responses to CMV specific

antigens (Oxford Diagnostic Laboratories, 2018). The test

purportedly has the potential to help transplant patients and

physicians manage immune regulated conditions. The

T-SPOT.CMV test leverages Oxford Immunotec’s proprietary

T-SPOT technology platform. The new test are performed at

Oxford Diagnostic Laboratories in Memphis, TN, where

validation was performed. The test results are available to

customers within 2 days of receipt of a whole blood sample.

This test was developed and its performance characteristics

determined by Oxford Diagnostic Laboratories. It has not

been cleared or approved by the FDA. Oxford Diagnostic

Laboratories is regulated under the Clinical Laboratory

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Improvement Amendments (CLIA) and the College of

American Pathologists (CAP) as an accredited laboratory to

perform high complexity clinical laboratory testing.

The measurement of CMV specific cellular immunity in organ

transplant recipients could contribute additional acuity to

serology based, CMV infection risk stratification, facilitating

optimization of immunosuppression and anti-viral prophylaxis

(Chanouzas, et al., 2018).

Chanouzas and colleagues (2018) conducted a pilot study of

renal transplant recipient (RTR's) responses in the

T-SPOT.CMV ELISPOT based assay. The investigators

recruited 108 RTR's 3 months post-transplantation,

immediately prior to the cessation of stratified anti-viral

prophylaxis, used in recipients from seropositive donors.

RTR's were monitored for CMV viremia and disease. Cellular

responses to peptides derived from CMV IE1 and pp65 were

measured, using the T-SPOT.CMV assay. At recruitment, no

CMV specific cellular immunity was detected by T-SPOT.CMV

in CMV seronegative recipients (IE1 ≤ 1spot / 2.5x105

PBMC's; pp65 ≤ 3 spots / 2.5x105 PBMC's). At recruitment,

CMV seropositive recipients who made a robust response to

both IE1 (>25 spots / 2.5x105 PBMC's) and pp65 (>50 spots /

2.5x105 PBMC's), were less likely to develop high level

viremia than those who responded to one or neither antigen

(0/28 vs 5/25; p<0.02). The authors concluded that, in CMV

seronegative RTR's, CMV specific cellular immunity measured

by T-SPOT.CMV was not detected prior to cessation of anti

viral prophylaxis. This differs from recent reports of CMV

specific cellular immunity in a proportion of CMV seronegative

RTR's, associated with protection from CMV infection. In

seropositive RTR's, a dual response to IE1 and pp65 at

recruitment, was associated with protection from subsequent

viremia. This suggests that assessing the diversity of response

to CMV antigens, may enhance risk stratification in this group.

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Cytomegalovirus (CMV) infection causes significant morbidity

and mortality after allogeneic hematopoietic cell

transplantation (allo-HCT). CMV management after HCT

includes risk stratification and mainly a preemptive strategy

with CMV viral load serial monitoring. Cell mediated immunity

(CMI) plays a role in CMV reactivation and can be assessed

by cytokine responses such as T cell production of interferon

gamma (IFN-γ). Quantification of CMV CMI may have value in

CMV management (Ariza-Heredia, et al., 2016).

Ariza-Heredia, et al. (2016) evaluated the potential of a CMV-

specific ELISPOT assay to determine CMI against CMV

reactivation in allo-HCT recipients ≤26 weeks post-HCT. This

is an ongoing, multi-center, prospective, observational study of

≥150 adult CMV seropositive allo-HCT recipients. CMV

management was according to institutional protocols. Date of

CMV reactivation, as defined by each institution, was

recorded. T cell responses were serially monitored pre-, and

every 2 weeks post-transplantation up to 26 weeks with an

ELISPOT assay that uses CMV-specific antigens IE-1 and

pp65 (T-SPOT.CMV, Oxford Diagnostics Laboratories®,

Memphis, TN). Data reviewed include patients reaching ≥12

weeks post-HCT by March 2016. Thirty-five patients across 6

sites reached ≥12 weeks post-HCT, and 15 reached ≥22

weeks. Majority of the patients were white (54%), males

(77%), with a median age of 57 (25–80) years, and had

unrelated (43%) or matched related (46%) HCT. CMV

reactivation occurred in 13 patients (37%) and time to first

reactivation occurred ≤10 weeks post-HCT. Average immune

response as measured by CMV-specific IE-1 and pp65 spot

counts (SPC) increased over time post-transplantation. The

preliminary analysis of the REACT study showed CMV

reactivation occurred early during transplantation when the

CMV immune response (measured by a CMV-specific

ELISPOT assay) was lower. In contrast, no reactivation was

seen later on when immune response was higher. This study

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may provide insights into the CMV immune response which

may guide personalized decisions regarding CMV

management.

Gamma Interferon Response

Chandrashekaran and colleagues (2018) noted that the

number of lung transplantations performed worldwide

continues to increase. There is a growing need in these

patients for more effective immunosuppressive medications

with less toxicity. These researchers summarized the recent

studies and developments in lung transplant

immunosuppression. Novel immunosuppressive medications

and strategies used in other solid organ transplantations were

being tried in lung transplantation. This included the use of co-

stimulation blockers like belatacept and mTOR inhibitors like

everolimus. Calcineurin sparing regimens have been

described in an attempt to minimize nephrotoxicity. Assays to

measure the bioactivity of immunosuppressive medications to

determine the global immune competence, such as

ImmuKnow assay and gamma interferon response are gaining

traction. The authors concluded that immunosuppression in

lung transplant is evolving with the development of newer

drugs and promising strategies to optimize

immunosuppression. These researchers stated that further

studies with multi-center randomized trials are needed to

increase the strength of the evidence.

Measurement of Donor-Derived Cell-Free DNA as a Biomarker for Allograft Rejection in Solid Organ Transplantation

Beck and colleagues (2015) stated that in solid organ

transplantation, sensitive real-time biomarkers to evaluate the

graft health are desirable to enable early intervention (e.g., to

avoid full-blown rejections). During rejection, high amounts of

graft-derived cell-free DNA (GcfDNA) are shed into the blood

stream. The quantification of this GcfDNA in

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allotransplantation is considered to fulfill this need, because it

can be measured with great precision and at reasonable cost.

In this study, patients from 2 ongoing studies in kidney (KTx)

and heart (HTx) transplantation were monitored blinded on a

scheduled basis, by means of a published universal droplet

digital PCR to quantify the GcfDNA. Immediately after

engraftment, GcfDNA reached high values (greater than 5 %

of total cfDNA), with a rapid decrease to values of less than

0.5 % within 1 week. Living-related KTx recipients showed

lower initial values, reflecting the absence of preservation

injury. Episodes of rejection in KTx and HTx were

accompanied by a significant increase of GcfDNA (greater

than 5-fold) above values in patients without complications,

occurring earlier than clinical or biochemical hints to rejection.

One case of rejection, which became clinically suspect after 1

year and was proven with biopsy, showed a significant 10-fold

increase 3 months earlier. The authors concluded that the

quantification of GcfDNA has the potential to detect rejection

episodes at early stages, when other means of diagnosis are

not effective. The method's non-invasiveness enables

monitoring of recipients at intervals that are desired to catch

rejections at early actionable stages to prevent full-blown

rejection. This biomarker will be particularly valuable in

regimens to minimize immunosuppression.

Sigdel and co-workers (2018) noted that standard non

invasive methods for detecting renal allograft rejection and

injury have poor sensitivity and specificity. Plasma donor-

derived cell-free DNA (dd-cfDNA) has been reported to

accurately detect allograft rejection and injury in transplant

recipients and shown to discriminate rejection from stable

organ function in KTx recipients. This study used a novel

single nucleotide polymorphism (SNP)-based massively

multiplexed PCR (mmPCR) methodology to measure dd

cfDNA in various types of KTx recipients for the detection of

allograft rejection/injury without prior knowledge of donor

genotypes. A total of 300 plasma samples (217 biopsy-

matched: 38 with active rejection (AR), 72 borderline rejection

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(BL), 82 with stable allografts (STA), and 25 with other injury

(OI)) were collected from 193 unique KTx patients; dd- cfDNA

was processed by mmPCR targeting 13,392 SNPs. Median dd

cfDNA was significantly higher in samples with biopsy- proven

AR (2.3 %) versus BL (0.6 %), OI (0.7 %), and STA (0.4 %) (p

< 0.0001 all comparisons). The SNP-based dd- cfDNA assay

discriminated active from non-rejection status with an area

under the curve (AUC) of 0.87, 88.7 % sensitivity (95 % CI: 77.7

to 99.8 %) and 72.6 % specificity (95 % CI: 65.4 to 79.8 %) at a

pre-specified cut-off (greater than 1 % dd- cfDNA). Of 13

patients with AR findings at a routine protocol biopsy 6-month

post-KTx, 12 (92 %) were detected positive by dd-cfDNA. This

SNP-based dd-cfDNA assay detected allograft rejection with

superior performance compared with the current standard of

care (SOC). The authors concluded that these data supported

the feasibility of using this assay to detect disease prior to renal

failure and optimize patient management in the case of allograft

injury. They stated that this rapid, accurate, and non-invasive

technology allows for detection of significant renal injury in

patients better than the current SOC, with the potential for better

patient management, more targeted biopsies, and improved

renal allograft function and survival.

The authors stated that a drawback of this study was that it

was a retrospective analysis of archived samples from a

single-center. However, the central geographical area

enabled all biopsies to be performed by a single pathologist,

which may have helped minimize variability in biopsy

classification; further, all experimenters were kept blinded

during the process of data generation. The retrospective study

design may have led to differences in patient characteristics

across the rejection groups (e.g., the STA group was enriched

with younger patients who may be better suited

immunologically to tolerate transplanted organs compared to

older-aged patients). However, these age differences likely

did not affect the validity of the study findings.

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Whitlam and associates (2019) stated that GcfDNA (donor

derived cell-free DNA) is an emerging marker of kidney

allograft injury. Studies examining the clinical validity of this

biomarker have previously used the graft fraction, or

proportion of total cell-free DNA that is graft-derived. These

researchers evaluated the diagnostic validity of absolute

measurements of GcfDNA, as well as calculated graft fraction,

for the diagnosis of graft dysfunction. Plasma GcfDNA, total

cell-free DNA, and graft fraction were correlated with biopsy

diagnosis as well as individual Banff scores. A total of 61

samples were included in the analysis. For the diagnosis of

antibody mediated rejection (AMR), the receiver-operator

characteristic (ROC) AUC of GcfDNA and graft fraction were

0.91 (95 % CI: 0.82 to 0.98) and 0.89 (95 % CI: 0.79 to 0.98),

respectively. Both measures did not diagnose borderline or

type 1A cellular mediated rejection (CMR). Graft fraction was

associated with a broader range of Banff lesions, including

lesions associated with CMR, while GcfDNA appeared more

specific for AMR. The authors concluded that the capacity for

absolute quantification, and lower barriers to implementation of

this methodology recommend it for further study. They stated

that the drawbacks of this study included a small sample size

and lack of a validation cohort.

Huang and co-workers (2019) noted that dd-cfDNA became

Medicare reimbursable in the United States in October 2017

for the detection of rejection in KTx recipients based on results

from its pivotal validation trial, but it has not yet been externally

validated. These researchers evaluated 63 adult KTx

recipients with suspicion of rejection with dd-cfDNA and

allograft biopsy. Of these, 27 (43 %) patients had donor-

specific antibodies (DSA) and 34 (54 %) were found to have

rejection by biopsy. The percentage of dd-cfDNA was higher

among patients with AMR (median 1.35 %; interquartile range

[IQR]: 1.10 % to 1.90 %) compared to those with no rejection

(median 0.38 %, IQR: 0.26 % to 1.10 %; p < 0.001) and CMR

(median: 0.27 %, IQR: 0.19 % to 1.30 %; p = 0.01). The dd

cfDNA test did not discriminate patients with CMR from those

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without rejection. The AUC for CMR was 0.42 (95 % CI: 0.17

to 0.66). For AMR, the AUC was 0.82 (95 % CI: 0.71 to 0.93)

and a dd-cfDNA greater than or equal to 0.74 % yielded a

sensitivity of 100 %, specificity 71.8 %, PPV 68.6 %, and NPV

100 %. The authors concluded that the dd-cfDNA test did not

discriminate CMR from no rejection among KTx recipients,

although performance characteristics were stronger for the

discrimination of AMR.

Khush and colleagues (2019) noted that standardized dd

cfDNA testing has been introduced into clinical use to monitor

KTx recipients for rejection. These investigators described the

performance of this dd-cfDNA assay to detect allograft

rejection in samples from HTx recipients undergoing

surveillance monitoring across the United States. Venous

blood was longitudinally sampled from 740 HT recipients from

26 centers and in a single-center cohort of 33 patients at high-

risk for AMR. Plasma dd-cfDNA was quantified by using

targeted amplification and sequencing of a SNP panel. The dd

cfDNA levels were correlated to paired events of biopsy- based

diagnosis of rejection. The median dd-cfDNA was 0.07

% in reference HTx recipients (2,164 samples) and 0.17 % in

samples classified as acute rejection (35 samples; p = 0.005).

At a 0.2 % threshold, dd-cfDNA had a 44 % sensitivity to

detect rejection and a 97 % NPV. In the cohort at risk for AMR

(11 samples), dd-cfDNA levels were elevated 3-fold in AMR

compared with patients without AMR (99 samples, p = 0.004).

The standardized dd-cfDNA test identified acute rejection in

samples from a broad population of HTx recipients. The

authors concluded that the reported test performance

characteristics will guide the next stage of clinical utility studies

of the dd-cfDNA assay.

Oellerich and co-workers (2019) noted that dd-cfDNA is a non

invasive biomarker for comprehensive monitoring of allograft

injury and rejection in KTx. dd-cfDNA quantification of

copies/ml plasma (dd-cfDNA[cp/ml]) was compared to dd

cfDNA fraction (dd-cfDNA[%]) at pre-specified visits in 189

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patients over 1 year post-KTx. In patients (N = 15, n = 22

samples) with biopsy-proven rejection (BPR), median dd

cfDNA(cp/ml) was 3.3-fold and median dd-cfDNA(%) 2.0-fold

higher (82 cp/ml; 0.57 %, respectively) than medians in Stable

Phase patients (N = 83, n = 408) without rejection (25 cp/ml;

0.29 %). Results for acute tubular necrosis (ATN) were not

significantly different from those with BPR. dd-cfDNA

identified unnecessary biopsies triggered by a rise in plasma

creatinine; ROC analysis showed superior performance (p =

0.02) of measuring dd-cfDNA(cp/ml) (AUC = 0.83) compared

to dd-cfDNA(%) (AUC = 0.73). Diagnostic odds ratios (DORs)

were 7.31 for dd-cfDNA(cp/ml), and 6.02 for dd-cfDNA(%) at

thresholds of 52 cp/ml and 0.43 %, respectively. Plasma

creatinine showed a low correlation (r = 0.37) with dd-cfDNA

(cp/ml). In a patient subset (N = 24) there was a significantly

higher rate of patients with elevated dd-cfDNA(cp/ml) with

lower tacrolimus levels (less than 8 μg/L) compared to the

group with higher tacrolimus concentrations (p = 0.0036)

suggesting that dd-cfDNA may detect inadequate

immunosuppression resulting in subclinical graft damage.

Absolute dd-cfDNA(cp/ml) allowed for better discrimination

than dd-cfDNA(%) of KTx patients with BPR and is useful to

avoid unnecessary biopsies.

The authors stated that drawbacks of this study included the

lack of protocol biopsies and the fact that the vast majority of

patients were of Caucasian origin. The findings from this study

support the role of dd‐cfDNA as a pivotal addition to the

methods currently used to achieve personalized

immunosuppression in KTx, such as immunological

monitoring, therapeutic drug monitoring, microbial screening,

and biopsy. Based on all this information, in agreement with

other reports, these researchers are confident that dd‐cfDNA

can be recommended for clinical translation as a valid tool to

aid personalized patient care for the benefit of transplanted

patients and healthcare payers. To achieve this, methods

providing a fast turn-around time, such as the ddPCR used

herein, will allow frequent monitoring and provide actionable

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results (e.g., to aid in decision‐making for biopsy).

CPT Codes / HCPCS Codes / ICD-10 Codes

Information in the [brackets] below has been added for clarification purposes. Codes requiring a 7th character are represented by "+":

Code Code Description

ImmuKnow Assay:

CPT codes covered if selection criteria are met:

86352 Cellular function assay involving stimulation

(eg, mitogen or antigen) and detection of

biomarker (EG, ATP)

Other CPT codes related to the CPB:

38240 Hematopoietic progenitor cell (HPC); allogeneic

transplantation per donor

ICD-10 codes covered if selection criteria are met:

A00.0 -

B99.9

Infectious and parasitic diseases

C00.0 -

D09.9

Malignant neoplasms [not covered for detection

of cellular immune function in individuals with

renal cell carcinoma]

T45.1X5+ Adverse effect of antineoplastic and

immunosuppressive drugs

ICD-10 codes not covered for indications listed in the CPB (not all-inclusive):

D47.z1 Post-transplant lymphoproliferative disorder

(PTLD)

D89.810 -

D89.813

Graft-versus-host disease

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K50.00 -

K50.019

Crohn's disease

K51.00 -

K51.919

Ulcerative colitis

M32.14 Glomerular disease in systemic lupus

erythematosus [Lupus nephritis]

T86.10 -

T86.819

T86.890 -

T86.899

Complications of transplanted organ

Z48.21 -

Z48.298

Aftercare following organ transplant

Z48.3 Aftercare following surgery for neoplasm

[monitoring the immune response following

surgery]

Z48.810 -

Z48.89

Encounter for other specified postprocedural

aftercare [monitoring the immune response

following surgery]

Z76.82 Awaiting organ transplant status

Other Transplantation Immune Cell Function Assay:

CPT codes not covered for indications listed in the CPB:

T-SPOT.CMV, gamma interferon response for measurement of the bioactivity of immunosuppressive medications in lung transplantation - no specific code:

0118U Transplantation medicine, quantification of

donor-derived cell-free DNA using whole

genome next-generation sequencing, plasma,

reported as percentage of donor-derived cell-

free DNA in the total cell-free DNA

ICD-10 codes not covered for indications listed in the CPB (not all-inclusive):

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T86.10 -

T86.49

T86.810 -

T86.819

T86.850 -

T86.99

Complications of transplanted organ

Z94.0 Kidney transplant status

Z94.1 Heart transplant status

Z94.2 Lung transplant status

Z94.3 Heart and lungs transplant status

Z94.4 Liver transplant status

Z94.83 Pancreas transplant status

The above policy is based on the following references:

1. 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.

2. Thai NL, Blisard D, Tom K, et al. Pancreas

transplantation under alemtuzumab (Campath-1H) and

tacrolimus: Correlation between low T-cell responses and

infection. Transplantation. 2006;82(12):1649-1652.

3. 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.

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4. Huang Y, Nizami Nazih Zuhdi I. Correlation of Cylex

ImmuKnow assay with lung allograft biopsy. J Heart Lung

Transplant. 2007;26:S175.

5. 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.

6. Sampson VB, Dunn SP, Rymeski B, et al. Failure of

immunosuppressive drug levels to predict T-cell reactivity

in pediatric transplant patients. J Pediatr Surg. 2008;43

(6):1134-1141.

7. Batal I, Zeevi A, Heider A, et al. Measurements of global

cell-mediated immunity in renal transplant recipients with

BK virus reactivation. Am J Clin Pathol. 2008;129(4):587

591.

8. Gupta S, Mitchell JD, Markham DW, et al. Utility of the

Cylex assay in cardiac transplant recipients. J Heart Lung

Transplant. 2008;27(8):817-822.

9. U.S. Food and Drug Administration (FDA). Cylex Immune

Cell Function Assay. 510(k) Summary. K013169.

Rockville, MD: FDA; April 2, 2002.

10. Cylex Inc. ImmuKnow [website]. Columbia, MD: Cylex;

2008. Available at:

http://www.cylex.net/index.html. Accessed October 28,

2008.

11. 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.

12. Indiana University School of Medicine. Investigation of

the Cylex ImmuKnow Assay. ClinicalTrials.gov Identifier

NCT00569842. Bethesda, MD: National Library of

Medicine; updated July 30, 2008.

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http://www.cylex.net/index.html

http://ClinicalTrials.gov


13. Cylex Inc. Cylex announces plans for multi-center trial of

ImmuKnow Assay in South Korea. Press Release.

Columbia, MD: Cylex; August 28, 2008.

14. Chon WJ, Brennan DC. Investigational methods in the

diagnosis of acute renal allograft rejection. UpToDate

[online serial]. Waltham, MA: UpToDate; October 2009.

15. Serban G, Whittaker V, Fan J, et al. Significance of

immune cell function monitoring in renal transplantation

after Thymoglobulin induction therapy. Hum Immunol.

2009;70(11):882-890.

16. 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.

17. 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.

18. Macedo C, Zeevi A, Bentlejewski C, et al. The impact of

EBV load on T-cell immunity in pediatric thoracic

transplant recipients. Transplantation. 2009;88(1):123

128.

19. Rossano JW, Denfield SW, Kim JJ, et al. Assessment of

the Cylex ImmuKnow cell function assay in pediatric

heart transplant patients. J Heart Lung Transplant.

2009;28(1):26-31.

20. Martinu T, Chen DF, Palmer SM. Acute rejection and

humoral sensitization in lung transplant recipients. Proc

Am Thorac Soc. 2009;6(1):54-65.

21. 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.

22. Bennett WM, Meyer L, Ridenour J, Batiuk TD.

Surveillance and modification of immunosuppression

minimizes BK virus nephropathy. Am J Nephrol. 2010;32

(1):10-12.

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23. 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.

24. Torío A, Fernández E, Montes-Ares O, et al. Lack of

association of immune cell function test with rejection in

kidney transplantation. Transplant Proc. 2011;43

(6):2168-2170.

25. De Paolis P, Favarò A, Piola A, et al. "Immuknow" to

measurement of cell-mediated immunity in renal

transplant recipients undergoing short-term evaluation.

Transplant Proc. 2011;43(4):1013-1016.

26. Huskey J, Gralla J, Wiseman AC. Single time point

immune function assay (ImmuKnow) testing does not aid

in the prediction of future opportunistic infections or acute

rejection. Clin J Am Soc Nephrol. 2011;6(2):423-429.

27. Cheng JW, Shi YH, Fan J, et al. An immune function

assay predicts post-transplant recurrence in patients with

hepatocellular carcinoma. J Cancer Res Clin Oncol.

2011;137(10):1445-1453.

28. Ling X, Xiong J, Liang W, et al. Can immune cell function

assay identify patients at risk of infection or rejection? A

meta-analysis. Transplantation. 2012;93(7):737-743.

29. Shino MY, Weigt SS, Saggar R, et al. Usefulness of

immune monitoring in lung transplantation using

adenosine triphosphate production in activated

lymphocytes. J Heart Lung Transplant. 2012;31(9):996

1002.

30. Rodrigo E, Lopez-Hoyos M, Corral M, et al. ImmuKnow(®)

as a diagnostic tool for predicting infection and acute

rejection in adult liver transplant recipients: Systematic

review and meta-analysis. Liver Transpl. 2012;18

(10):1245-1253.

31. Akimoto M, Yunoue S, Otsubo H, et al. Assessment of

peripheral blood CD4+ adenosine triphosphate activity in

patients with rheumatoid arthritis. Mod Rheumatol.

2013;23(1):19-27.

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32. Lopez-Hoyos M, Rodrigo E, Arias M. The usefulness of

intracellular adenosine-5'-triphosphate measurement in

CD4+ cells in renal transplant. Nefrologia. 2013;33

(3):381-388.

33. Israeli M, Klein T, Herscovici C, et al. Cellular immune

function monitoring after allogeneic haematopoietic cell

transplantation: Evaluation of a new assay. Clin Exp

Immunol. 2013;172(3):475-482.

34. Brandhorst G, Weigand S, Eberle C, et al. CD4+ immune

response as a potential biomarker of patient reported

inflammatory bowel disease (IBD) activity. Clin Chim

Acta. 2013;421:31-33.

35. Wong MS, Boucek R, Kemna M, et al. Immune cell

function assay in pediatric heart transplant recipients.

Pediatr Transplant. 2014;18(5):485-490.

36. Ryan CM, Chaudhuri A, Concepcion W, Grimm PC.

Immune cell function assay does not identify biopsy-

proven pediatric renal allograft rejection or infection.

Pediatr Transplant. 2014;18(5):446-452.

37. Chon WJ, Brennan DC. Investigational methods in the

diagnosis of acute renal allograft rejection. UpToDate

[online serial]. Waltham, MA: UpToDate; reviewed July

2014.

38. Liu J, Pan Y, Tang LJ, et al. Low adenosine triphosphate

activity in CD4+ cells predicts infection in patients with

lupus nephritis. Clin Exp Rheumatol. 2014;32(3):383-389.

39. Ravaioli M, Neri F, Lazzarotto T, Bertuzzo VR, et al.

Immunosuppression modifications based on an

immune response assay: Results of a randomized,

controlled trial. Transplantation. 2015;99(8):1625-1632.

40. Andreotti C, Zortea M, Provenzani A, et al. Immuknow

and long term kidney graft. G Ital Nefrol. 2015;32(2).

41. Jwa E, Hwang S, Kwon YJ, et al. In vitro immune cell

monitoring as a guide for long-term

immunosuppression in adult liver transplant

recipients. Korean J Hepatobiliary Pancreat Surg.

2015;19(4):139-148.

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42. Zheng K, Zhang JP, Tan JM, et al. Lack of clinical

significance of the ImmuKnow(TM)-Cylex assay for the

detection of cellular immune function in patients with

renal cell carcinoma. Genet Mol Res. 2015;14(3):11543-

11550.

43. Maki K, Takeno S, Aisu N, et al. CD4+ T-lymphocytes

are activated by surgical stress following colorectal

resection in cancer patients. Mol Clin Oncol. 2015;3

(1):79-82.

44. Zhang W, Zhong H, Zhuang L, et al. Peripheral blood

CD4(+) cell ATP activity measurement to predict HCC

recurrence post-DCD liver transplant. Int J Clin Pract.

2016;70 Suppl 185:11-16.

45. Sindhi R, Ashokkumar C, Higgs BW, et al. Profile of the

Pleximmune blood test for transplant rejection risk

prediction. Expert Rev Mol Diagn. 2016;16(4):387-393.

46. Ashokkumar C, Soltys K, Mazariegos G, et al. Predicting

cellular rejection with a cell-based assay: Preclinical

evaluation in children. Transplantation. 2017;101

(1):131-140.

47. Chiereghin A, Petrisli E, Ravaioli M, et al. Infectious

agents after liver transplant: Etiology, timeline and

patients' cell-mediated immunity responses. Med

Microbiol Immunol. 2017;206(1):63-71.

48. Schmidt T, Schub D, Wolf M, et al. Comparative

analysis of assays for detection of cell-mediated

immunity toward cytomegalovirus and M. tuberculosis

in samples from deceased organ donors. Am J

Transplant. 2014;14(9):2159-2167.

49. Luo Y, Ji WB, Duan WD, et al. Delayed introduction of

immunosuppressive regimens in critically ill patients

after liver transplantation. Hepatobiliary Pancreat Dis

Int. 2017;16(5):487-492.

50. Chanouzas D, Small A, Borrows R, Ball S. Assessment

of the T-SPOT.CMV interferon-γ release assay in renal

transplant recipients: A single center cohort study.

PLoS One. 2018;13(3):e0193968.

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51. Ariza-Heredia E, Shah DP, El Chaer F, et al. Prospective

observational study to evaluate a cytomegalovirus

(CMV)–specific T-SPOT assay in hematopoietic stem

cell transplant recipients: The REACT Study interim

data review. Open Forum Infect Dis. 2016; 3 (Suppl 1):

2297.

52. Oxford Diagnostic Laboratories. The T-Spot.CMV test

[website]. Abingdon, UK: Oxford Immunotec; 2018.

Available at:

http://www.oxforddiagnosticlabs.com/products-and-

services/t-spot-cmv/. Accessed August 19, 2018.

53. Chandrashekaran S, Crow Pharm SA, Shah SZ, et al.

Immunosuppression for lung transplantation: Current

and future. Curr Transplant Rep. 2018;5(3):212-219.

54. Beck J, Oellerich M, Schulz U, et al. Donor-derived cell-

free DNA Is a novel universal biomarker for allograft

rejection in solid organ transplantation. Transplant

Proc. 2015;47(8):2400-2403.

55. Sigdel TK, Archila FA, Constantin T, et al. Optimizing

detection of kidney transplant injury by assessment of

donor-derived cell-free DNA via massively multiplex

PCR. J Clin Med. 2018;8(1).

56. Whitlam JB, Ling L, Skene A, et al. Diagnostic

application of kidney allograft-derived absolute cell-

free DNA levels during transplant dysfunction. Am J

Transplant. 2019;19(4):1037-1049.

57. Huang E, Sethi S, Peng A, et al. Early clinical experience

using donor-derived cell-free DNA to detect rejection

in kidney transplant recipients. Am J Transplant.

2019;19(6):1663-1670.

58. Khush KK, Patel J, Pinney S, et al. Noninvasive

detection of graft injury after heart transplant using

donor-derived cell-free DNA: A prospective multicenter

study. Am J Transplant. 2019 Mar 5 [Epub ahead of

print].

59. Oellerich M, Shipkova M, Asendorf T, et al. Absolute

quantification of donor-derived cell-free DNA as a

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http://www.oxforddiagnosticlabs.com/products-and-


marker of rejection and graft injury in kidney

transplantation: Results from a prospective

observational study. Am J Transplant. 2019 May 7

[Epub ahead of print].

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Copyright Aetna Inc. All rights reserved. Clinical Policy Bulletins are developed by Aetna to assist in administering plan

benefits and constitute neither offers of coverage nor medical advice. This Clinical Policy Bulletin contains only a partial,

general description of plan or program benefits and does not constitute a contract. Aetna does not provide health care

services and, therefore, cannot guarantee any results or outcomes. Participating providers are independent contractors

in private practice and are neither employees nor agents of Aetna or its affiliates. Treating providers are solely

responsible for medical advice and treatment of members. This Clinical Policy Bulletin may be updated and therefore is

subject to change.

Copyright © 2001-2020 Aetna Inc.

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AETNA BETTER HEALTH® OF PENNSYLVANIA

Amendment to Aetna Clinical Policy Bulletin Number: 0773 Transplant

Immune Cell Function Assays

There are no amendments for Medicaid.

www.aetnabetterhealth.com/pennsylvania annual 10/01/2020

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http://www.aetnabetterhealth.com/pennsylvania

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