Università degli Studi di Roma Tor Vergata
The new system for the quality control in HPC units before the reinfusion in autologous transplant Maria Cristina Scerpa (1), Nicola Daniele (2), Cecilia Rossi (2), Giancarlo Isacchi (2), Francesco Zinno (2) (1)Tor Vergata University and Bambino Gesù Pediatric Hospital (Rome, IT); (2)Tor Vergata University and Bambino Gesù Pediatric Hospital (Rome, IT)
OBJECTIVE The crucial points of autologous stem cell transplantation are represented by the cryopreservation, the storage and the reinfusion of these units. In our center, the cell viability, the integrity of the bag and the hematopoietic progenitors clonogenic assay were evaluated before the reinfusion of HPC-A. This quality control should be made 14 days before the reinfusion of HPC-A to have the result of the functional test on the proliferative capacity of haematopoietic progenitors. To fulfill the clinical requirements, it is not always possible to meet these deadlines. Therefore, this study was designed to assess the potential of the Nucleocounter NC-3000 (Chemometec, Denmark) in our clinical routine. This device allows a rapid assessment of cell functionality by determining the intracellular levels of reduced glutathione (GSH) and mitochondrial transmembrane potential.
METHODS
PATIENTS The study was conducted on 75 vials control were carried out on HPC-A from 46 patients AGE (years)
SEX M/F
DIAGNOSIS
3
5
1/2
Neuroblastoma
2
45
M
Mantle cell lymphoma Medulloblastoma
NUCLEOCOUNTER NC3000
measurement of the cellular level of reduced thiols NADP+
NADPH + H+
Glu-Cys-Gly
2 Glu-Cys-Gly -S-H
1/2
B)
ISHAGE PROTOCOL
-S-S-
4,5
A) CYTOFLUOROMETRY FACS CALIBUR
N° ptz
3
MEASUREMENT OF CELL VIABILITY
Glu-Cys-Gly
1
2
M
Mucopolysaccharidosis
2
12
M
Rhabdomyosarcoma
7
17
4/3
Non Hodgkin Lymphoma
mitochondrial membrane potential detection
11
30
7/4
Hodgkin Lymphoma
JC-1 assay
1
11
M
Retinoblstoma
3
11
M
Acute Myeloid Leukemia
7
60
3/4
Multiple Myeloma
2
5
1/1
Thalassemia
2
6
F
Germinal Tumor
2
2
M
Glioblastoma
Glutathione reductase
C) POTENTIAL PROLIFERATIVE OF THE HEMATOPOIETIC PROGENITORS CFU-GM
HSC
BFU-E
Clonogenic tests for Hematopoietic progenitors
CFU-GEMM
HPC-A
RESULTS JC-1 vs Potential Proliferative of Hematopoietic Cells
JC-1 vs Viability of CD34+ cells
700 80,00
Necrotic cells 70,00
500 400 300
Healthy
PI Negative cells 200
cells
r= - 0.43
100 0 0,00
10,00
20,00
30,00
40,00
50,00
60,00
70,00
80,00
JC-1
-100
% viability of CD34+ cells
Number of colonies
600
60,00
50,00
40,00
30,00
20,00
r=- 0.69 10,00
0,00 0
CUTT OFF JC-1=30
20
40
60
JC-1<30
350
P<0,0001
100
120
JC-1
% of JC-1
JC-1>30
80
% Viabilty of GSH vs % Viability CD45+ cells
Number of Colonies
90
300
Viability of CD34+ cells vs Potential Proliferative Hematopoitic cells
140
80 70
200
150
100
120
60 50 40 30 20
50
r=0.58
10 0
%JC-1
Number of Colonies
%JC-1
% Viabilty of CD34+ cells
% Viability of GSH
Number of cells
P<0,0001 250
100
80
60
40
r= 0,50 20
0
Number of Colonies
0
10
20
30
40
50
60
70
80
90
100
% Viability of CD45+cells
The evaluation of cells in pre-apoptosis measured by JC-1 assay showed a negative correlation (r = - 0,43) with the total number of colonies obtained after seeding in Methocult H4434 cell life of 50,000/ml. In this study, we observed a statistically significant difference (p = <0.0001) comparing the median number of colonies obtained with a value of JC-1 <30% ( 46.86; SD 58.07) to the number of colonies obtained with a value of JC-1>30% vs (171.14; SD 145.10).
The comparison between the flow cytometry method and functional assays with NucleoCounter showed a positive correlation between the percentage of cells CD45 + / CD34 + / 7AAD- and the percentage of viable cells in the GSH assay (r=0,58).
0 0
100
200
300
400
500
600
700
Number of colonies
The cellular function determined by the assessment of the percentage of JC-1 was compared with the flow cytometry method on same group of samples. The results have shown a negative correlation between the viable CD34+ cells and the % of JC-1(r =-0.69). Moreover, we observed a positive correlation between the median number of colonies obtained with the percentage of CD34+/CD45+/7AAD- cells obtained with the ISHAGE protocol.
CONCLUSIONS The use of functional assays of reduced glutathione and of the number of pre-apoptosis cells measured by NucleoCounter allows a rapid evaluation on multiple samples about the functional capacity of the cells which must be infused to the patients. The quality control performed before the infusion of stem cells, especially in the autologous transplant, would require the clonogenic assay that is the actually only test of cell function. The main lack is the need to have the quality control of the vitality of CD45+/CD34+ cells and the need to perform the clonogenic assay about 14 days before transplantation. In this study, we observed a correlation between the clonogenic assay and tests that allow the assessment of cell apoptosis. In conclusion, we suggest to divide samples of HPC-A in which the quality control showed a value of JC-1>30% from samples in which the quality control showed a value of JC-1<30% , so that the clonogenic assay can be performed only in cases with an established cut-off. Maria Cristina Scerpa - SIMT, IRCCS Bambino Gesù Pediatric Hospital - Email: mcscerpa@yahoo.it