PMID- 10861713
OWN - NLM
STAT- MEDLINE
DCOM- 20000728
LR  - 20190905
IS  - 0197-3851 (Print)
IS  - 0197-3851 (Linking)
VI  - 20
IP  - 6
DP  - 2000 Jun
TI  - Quantitative FISH analysis and in vitro suspension cultures of erythroid cells 
      from maternal peripheral blood for the isolation of fetal cells.
PG  - 479-86
AB  - Several techniques for the enrichment of nucleated fetal red blood cells present 
      in maternal blood have been reported. Here we describe the use of a quantitative 
      fluorescence in situ hybridization (FISH) method and in vitro suspension cultures 
      of erythroid cells from newborn cord blood and maternal peripheral blood. 
      Together with a rapid high performance liquid chromatography (HPLC) method, that 
      allows us to determine as few as 100 cells containing haemoglobin F (HbF), we 
      have scrutinized the reported enrichment methods for fetal nucleated cells in 
      peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood 
      were performed. The method comprises a cell lysis method for depletion of red 
      cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 
      cells each) on microscopic slides, and inclusion of internal controls to monitor 
      the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor 
      cells from maternal peripheral blood were analysed for the expansion of fetal 
      cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of 
      peripheral blood from 26 pregnant women were grown in media containing growth 
      factors and hormones to yield over 10(7) of immature erythroid cells within two 
      weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A 
      total of approximately 8x10(8) maternal cells were added into tissue culture 
      medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid 
      cells after two weeks. Whereas fetal cells, alone or added into cultures of 
      peripheral blood, grow rapidly and can be detected quantitatively, we could not 
      find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) 
      peripheral blood cells probed by FISH analysis (half of which were from 
      pregnancies with male fetuses) no single Y chromosome was detected. In summary, 
      suspension cultures of erythroid cells can be established routinely and easily. 
      With the quantitative FISH technique used, 750 000 cells per slide can be 
      screened reliably for cells with Y chromosomes. However, the stringent 
      quality-criteria and most elaborate methods indicate that fetal cells in maternal 
      peripheral blood can not be found using the current technology.
CI  - Copyright 2000 John Wiley & Sons, Ltd.
FAU - Huber, K
AU  - Huber K
AD  - Ludwig Boltzmann Inst. f. molekulargenetische Laboratoriumsdiagnostik, 
      Donauspital SMZ-Ost, Vienna, Austria.
FAU - Bittner, J
AU  - Bittner J
FAU - Worofka, B
AU  - Worofka B
FAU - Rosen, A
AU  - Rosen A
FAU - Hafner, E
AU  - Hafner E
FAU - Philipp, K
AU  - Philipp K
FAU - Bauer, K
AU  - Bauer K
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Prenat Diagn
JT  - Prenatal diagnosis
JID - 8106540
RN  - 9034-63-3 (Fetal Hemoglobin)
SB  - IM
MH  - Cell Culture Techniques
MH  - Cell Separation/*methods
MH  - Chromatography, High Pressure Liquid
MH  - Erythroid Precursor Cells/*cytology
MH  - Female
MH  - Fetal Blood/*cytology
MH  - Fetal Hemoglobin/analysis
MH  - Flow Cytometry
MH  - Humans
MH  - *In Situ Hybridization, Fluorescence
MH  - Male
MH  - Polymerase Chain Reaction
MH  - Pregnancy
EDAT- 2000/06/22 10:00
MHDA- 2000/08/06 11:00
CRDT- 2000/06/22 10:00
PHST- 2000/06/22 10:00 [pubmed]
PHST- 2000/08/06 11:00 [medline]
PHST- 2000/06/22 10:00 [entrez]
AID - 10.1002/1097-0223(200006)20:6<479::AID-PD861>3.0.CO;2-8 [pii]
AID - 10.1002/1097-0223(200006)20:6<479::aid-pd861>3.0.co;2-8 [doi]
PST - ppublish
SO  - Prenat Diagn. 2000 Jun;20(6):479-86. doi: 
      10.1002/1097-0223(200006)20:6<479::aid-pd861>3.0.co;2-8.