1. The study measured intracellular ATP levels in stimulated CD4+ T lymphocytes from 40 stable pediatric liver transplant patients to assess immune system function and guide immunosuppressive therapy. 2. ATP levels were similar to adult values, ranging from 200-400 ng/mL on average. 3. Patients on monotherapy of calcineurin inhibitors cyclosporine or tacrolimus had significantly higher ATP levels than those on double therapy with mycophenolate mofetil, indicating stronger immunosuppression with monotherapy.

1. Clinical Value of a Single Determination of Intracellular ATP Levels in
Stimulated CD4؉ T Lymphocytes in Pediatric Patients With Stable
Liver Transplantation
M. Serrano, J.C. Meneu, E. Medina, F.J. Alfaro, J.A. Martinez-Flores, S. Mora, J.M. Morales,
E. Paz Artal, J. Manzanares, and A. Serrano
ABSTRACT
In the follow-up of transplanted patients under immunosupression, the functional
assessment of CD4ϩ T cells activation by measuring intracellular ATP levels in vitro, using
the Immuknow test give information on how immune system is functioning. Therefore, it
has been reported that low levels of ATP correlate with the risk of opportunistic infection.
Although limited, comprehensive results in adult recipients as well as in pediatric
transplanted patients have been reported. Forty stable liver pediatric transplanted patients
(mean age: 11.0 years [SD 5.65]), within at least 1 year of transplant were selected for a
scheduled review, and an unique determination of Immuknow was performed. Average
ATP levels were 317 ng/mL (200–400 ng/mL) which were similar to the values observed
in adult population. ATP values among patients with monotherapy Cyclosporin A (CSA)
or tacrolimus (TAC) were significantly higher (P ϭ .005) than in patients with double
immunosupressive therapy using either CSA/TAC ϩ Mycophenolate Mofetil (MMF). In
CSA treatment, there are significant differences (P ϭ .0003) between monotherapy and
double therapy, but in the case of TAC we did not find differences (P Ͼ .1). A single
determination of levels of ATP on CD4ϩ lymphocytes, can provide additional information
that could be used as a complementary test to guide immunosuppressive therapy in
paediatric liver transplant recipients.
LIVER transplant patients must maintain a chronic
immunosuppressive therapy usually with calcineurin
inhibitors (CNI) to prevent allograft rejection.1
Optimal
treatment must be maintained within a narrow therapeutic
range, because if there is excessive inmunosupression, there
is a great risk of opportunistic infections. On the other
hand, insufficient immunosupression would not be effective
to prevent rejection. Usually pediatric liver transplant re-
cipients are treated with CNI, such as cyclosporine (CsA) or
tacrolimus (TAC), in monotherapy, or in combination with
mycophenolate mofetil (MMF) (double therapy).2
During
follow-up, drug plasma levels of CNI and liver enzymed
values are monitored, but this is not enough to provide
functional data on the degree of immunosuppression.3
Functional assessment of CD4 ϩ T cells by measuring
intracellular ATP levels after stimulation in vitro using
Immuknow allows a better knowledge of the state of
lymphocyte activity, and seems to correlate better with the
clinic status.3
Its efficacy has already been demonstrated for
monitoring immunosupression.4
Comprehensive results in
adult recipients have been reported as well as in pediatric
transplanted patients, but they are very limited.5
There is
controversy about the optimum ATP concentration, de-
pending on age,6
needed to monitor graft evolution. The
aim of the present study is to know the clinical value of a
single determination of intracellular ATP levels after stim-
ulation in CD4 ϩ lymphocytes in pediatric patients with a
liver transplantation.
From the Servicio inmunología (M.S., F.J.A., J.A.M.-F., S.M.,
E.P.A., A.S.), Gastroenterología y Hepatología Pediátrica (E.M.,
J.M.), Servicio Cirugía Gral. Digestivo y Trasplantes Abdomina-
les (J.C.M.), Servicio Nefrología (J.M.M.), Hospital Universitario
“12 de Octubre,” Madrid, Spain.
Address correspondence to Manuel Serrano Immunology De-
partment Hospital 12 de Octubre Avda. Córdoba s/n 28041
Madrid, Spain. E-mail: mserranobl@gmail.com
0041-1345/12/$–see front matter © 2012 by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.transproceed.2012.09.080 360 Park Avenue South, New York, NY 10010-1710
2622 Transplantation Proceedings, 44, 2622–2624 (2012)

2. PATIENTS AND METHODS
Patients were selected from a scheduled review. The resulting popu-
lation were 40 stable liver pediatric transplanted patients, aged 11
years (SD 5.65), with at least 1 year transplant time, and without
rejection and infection (viral or bacterial) in the previous 6 and 4
months, respectively. Mean age at transplantation was 3.56 years (SD ϭ
4.2), and time from transplantation at the moment of the study was
7.67 years (SD ϭ 4.9). Based on immunosuppressive treatment, two
groups were defined: patients treated with cyclosporine (CSA), 12/40
(30%), and patients treated with tacrolimus (TAC), 28/40 (70%).
Monotherapy or double therapy with mycophenolate mofetil (MMF)
was also considered. We determine a single measure to quantify the
ATP levels of in CD4ϩ lymphocytes using the Cylex Immuknow test.
RESULTS
Average ATP levels were 317 ng/mL (SD ϭ 141.2). ATP
values among patients with monotherapy (CSA or TAC) were
significantly higher (P ϭ .005) than in patients with double
therapy (CSA/TAC ϩ MMF), values were 359.28 Ϯ 158.38
ng/mL, and 251.25 Ϯ 67.67 ng/mL, respectively (Table 1).
Furthermore, in CSA treatment, there are significant differ-
ences (P ϭ .0003) between montherapy and double therapy
patients (Fig 1) but in the case of TAC we did not find
differences (P Ͼ .1) in the same groups (Fig 1). Although there
were no significant differences, the dispersion of the results is
higher in patients with Tac (334.35 Ϯ 163.47 ng/mL) than
patients with CSA (459 Ϯ 89.65 ng/mL) with a tendency to
greater immunosuppression in the TAC group (Fig 1).
There were no statistically significant differences (P Ͼ
0.1) in different age groups, 227.2 Ϯ 44.18 ng/mL and
335.94 Ϯ 24.55 ng/mL in younger and older patients than 4
years, respectively (Table 1). We found no differences in
other age ranges (data not shown). We found no statistically
significant correlation between plasmatic levels of Tac
(mean: 4.28 Ϯ 0.72 ng/dL) or CyA (mean: 124.5 Ϯ 39.6
ng/dL) and ATP levels (correlation coefficient Ϫ0.004, P ϭ
.982 and Ϫ0.005, P ϭ .986, respectively).
DISCUSSION
In our study ATP levels of pediatric patients with stable liver
transplant would range from 200 ng/mL to 400 ng/mL similar to
adult transplant recipients. The mean value of intracellular ATP
levels in patients treated with TAC was closer to the expected
values for a correct immunosuppression than those treated with
CSA. In addition, CSA needed to be combined with another drug
(MMF) to reach standard normal values.
Administration of TAC as monotherapy or in combina-
tion with MMF shows similar results in ATP levels. These
results suggest that use of TAC as monotherapy can be
useful as an optimal immunossuppresive in this pediatric
population. Consequently, in some it might be possible to
avoid the MMF administration, minimizing the side effects
of the double therapy.
As in other studies, we found no differences in ATP levels
between different age groups, so in the future perhaps it will
not be necessary to make different age-groups,7,8
Other
markers of clinical status, such as viral load, drug plasma
levels,7,8
or liver function tests do not correlate with the
immune function status. This study has several limitations:
Fig 1. ATP levels (ng/mL) on the
CD4 lymphocytes if different treat-
ment groups; CSA, cyclosporine;
TAC, tacrolimus; D-CSA, cyclo-
sporine double therapy; D-TAC,
Tacrolimus double therapy.
Table 1. Group ATP Levels
Group
Patient
Number ATP levels (ng/mL)
Age Ͻ 4 years 6 227,2 Ϯ 44,2 (151–351)
Age Ͼ 4 years 34 335,9 Ϯ 24,5 (150–883)
Tac (mono/double
therapy)
28 334,3 Ϯ 163,47 (150–542)
CsA (mono/double
therapy)
12 459,0 Ϯ 89,65 (361–560)
Tac/CsA monotherapy 27 359,3 Ϯ 158,4 (293,9–424,65)
CsA/Tac double therapy 13 251,2 Ϯ 67.7 (208,25–294,25)
Note: Total patients ϭ 40; age average ϭ 11.0 years; age range ϭ 2.2–21.6
years.
SINGLE DETERMINATION OF INTRACELLULAR ATP 2623

3. the small sample of patients and the possible bias using only
one determination of ATP levels.
In summary, ATP levels of pediatric patients with stable
liver transplant would range from 200 ng/mL to 400 ng/mL.
ATP levels are similar in adults and pediatric patients. A
single determination of intracellular ATP levels in CD4 ϩ
lymphocytes could be useful indicating the level of immu-
nosuppression. However, to improve the immune response
evaluation efficacy it should be used in combination with
other immunosupressor biomarkers.
ACKNOWLEDGEMENTS
We thank Antonia Valero Pavón, Pilar Suárez Olivar and Marga-
rita Sevilla Sánchez for their excellent technical assistance.
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 82:663, 2006
2. Kelly DA. Long-term challenges of immunosuppression in
pediatric patients. Transplant Proc 37:1657, 2005
3. Kowalski R, Post D, Schneider MC, et al: Immune cell
function testing: an adjunct to therapeutic drug monitoring in
transplant patient management. Clin Transplant 17:77, 2003
4. Torio A, Fernandez EJ, Montes-Ares O, et al: Lack of
association of immune cell function test with rejection in kidney
transplantation. Transplant Proc 43:2168, 2011
5. Millan O, Sanchez-Fueyo A, Rimola A, et al: Is the intracel-
lular ATP concentration of CD4ϩ T-Cells a predictive biomarker
of immune status in stable transplant recipients? Transplantation
88:S78, 2009
6. Comans-Bitter WM, de Groot R, van den Beemd R, et al:
Immunophenotyping of blood lymphocytes in childhood. Refer-
ence values for lymphocyte subpopulations. J Pediatr 130:388, 1997
7. Israeli M, Klein T, Sredni B, et al: ImmuKnow: a new
parameter in immune monitoring of pediatric liver transplantation
recipients. Liver Transpl 14:893, 2008
8. Hooper E, Hawkins DM, Kowalski RJ, et al: Establishing
pediatric immune response zones using the Cylex ImmuKnow
assay. Clin Transplant, 2005
2624 SERRANO, MENEU, MEDINA ET AL