Pitfalls in detection of contaminating neuroblastoma cells by tyrosine hydroxylase RT-PCR due to catecholamine-producing hematopoietic cells.
BACKGROUND
RT-PCR analysis of compounds of catecholamine metabolism (in particular tyrosine hydroxylase, TH) is widely used for the detection of contaminating neuroblastoma cells in hematopoietic stem cell preparations. Due to reports in the literature showing that hematopoietic cells are also able to produce catecholamines, we investigated whether TH-RT-PCR is really suitable for this purpose.


METHODS
Besides neuroblastoma cells, mononuclear blood cells, apheresis preparations and hematopoietic stem cells were used for single and nested RT-PCR. In addition to TH, the expressions of dopamine-beta-hydroxylase and noradrenaline transporter were analyzed.


RESULTS
Using single RT-PCR, a clear discrimination between neuroblastoma and hematopoietic cells was possible. However, by using nested RT-PCR, the "neuroblastoma markers" were also detected in a significant percentage of non-mobilized mononuclear blood cells, in mononuclear blood cells of healthy donors mobilized with G-CSF, and in highly purified CD34+ and CD133+ stem cells from healthy mobilized donors.


CONCLUSIONS
Our results raise the question of whether the RT-PCR analysis of compounds of catecholamine metabolism is suitable and selective enough to detect the contamination of hematopoietic stem cells by a low number of neuroblastoma cells.
Kuçi
Zyrafete
Z
Department of Hematology and Oncology, Children's University Hospital, D-72076 Tübingen, Germany.
Seitz
Gabriele
G
Kuçi
Selim
S
Kreyenberg
Hermann
H
Schumm
Michael
M
Lang
Peter
P
Niethammer
Dietrich
D
Handgretinger
Rupert
R
Bruchelt
Gernot
G
D002395:Catecholamines / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D004299:Dopamine beta-Hydroxylase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D020869:Gene Expression Profiling; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics
D002395:Catecholamines; D014446:Tyrosine 3-Monooxygenase; D004299:Dopamine beta-Hydroxylase