PMID- 10213199
OWN - NLM
STAT- MEDLINE
DCOM- 19990628
LR  - 20191103
IS  - 0196-4763 (Print)
IS  - 0196-4763 (Linking)
VI  - 35
IP  - 4
DP  - 1999 Apr 1
TI  - Fetal erythroblast isolation up to purity from cord blood and their culture in 
      vitro.
PG  - 337-45
AB  - BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for 
      noninvasive prenatal genetic investigation. We previously showed that human 
      erythroblasts can be recovered from bone marrow and blood bank buffy coats by a 
      physical cell separation. In the present study, we modified our previous 
      methodology, taking into account the peculiar behavior of erythroblasts in 
      response to modifications of pH and osmolality of the separation medium. METHODS: 
      Twenty to forty milliters of cord blood were initially centrifuged on 
      Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a 
      continuous density gradient (1.040-1.085 g/ml). Two different gradients were 
      initially compared: the first was iso-osmolar and neutral, whereas the second 
      also contained an ionic strength gradient and a pH gradient (triple gradient). A 
      subsequent monocyte depletion was performed by using magnetic microbeads coated 
      with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by 
      sedimentation velocity. Purified cells were investigated by analyses with 
      fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization 
      (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were 
      cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and 
      neutral continuous density gradient, two separated bands of nucleated red blood 
      cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy 
      fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple 
      gradient, NRBCs were shifted to the low-density region. Monocyte depletion by 
      immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast 
      population. FACS and FISH analyses and immunocytochemistry substantiated the 
      purity of the isolated cell fraction, which was successfully cultured in vitro. 
      CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to 
      homogeneity from cord blood, but further refinements of the isolation procedure 
      are necessary before the same results can be obtained from maternal peripheral 
      blood.
FAU - Sitar, G
AU  - Sitar G
AD  - Clinica Medica 2, IRCCS Policlinico S. Matteo, Universita degli Studi di Pavia, 
      Italy.
FAU - Garagna, S
AU  - Garagna S
FAU - Zuccotti, M
AU  - Zuccotti M
FAU - Falcinelli, C
AU  - Falcinelli C
FAU - Montanari, L
AU  - Montanari L
FAU - Alfei, A
AU  - Alfei A
FAU - Ippoliti, G
AU  - Ippoliti G
FAU - Redi, C A
AU  - Redi CA
FAU - Moratti, R
AU  - Moratti R
FAU - Ascari, E
AU  - Ascari E
FAU - Forabosco, A
AU  - Forabosco A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Cytometry
JT  - Cytometry
JID - 8102328
SB  - IM
MH  - Blood Sedimentation
MH  - Cells, Cultured
MH  - Centrifugation, Density Gradient
MH  - Erythroblasts/*cytology
MH  - Fetal Blood/*cytology
MH  - Humans
MH  - Leukocytes, Mononuclear
MH  - Sex Chromosomes
EDAT- 1999/04/23 02:03
MHDA- 2000/06/20 09:00
CRDT- 1999/04/23 02:03
PHST- 1999/04/23 02:03 [pubmed]
PHST- 2000/06/20 09:00 [medline]
PHST- 1999/04/23 02:03 [entrez]
AID - 10.1002/(SICI)1097-0320(19990401)35:4<337::AID-CYTO6>3.0.CO;2-U [pii]
AID - 10.1002/(sici)1097-0320(19990401)35:4<337::aid-cyto6>3.3.co;2-l [doi]
PST - ppublish
SO  - Cytometry. 1999 Apr 1;35(4):337-45. doi: 
      10.1002/(sici)1097-0320(19990401)35:4<337::aid-cyto6>3.3.co;2-l.