PMID- 8875053
OWN - NLM
STAT- MEDLINE
DCOM- 19970218
LR  - 20161123
IS  - 0196-4763 (Print)
IS  - 0196-4763 (Linking)
VI  - 25
IP  - 1
DP  - 1996 Sep 1
TI  - Improved fetal nucleated erythrocyte sorting purity using intracellular antifetal 
      hemoglobin and Hoechst 33342.
PG  - 37-45
AB  - Fetal nucleated erythrocytes (FNRBC) flow sorted from maternal peripheral blood, 
      using monoclonal antibodies (mAb) that bind fetal cell surface antigens, are a 
      noninvasive source of fetal DNA for prenatal diagnosis. These mAbs, however, also 
      bind antigens shared by maternal cells. In sorted populations, this results in 
      maternal cell contamination and low fetal cell purities, which complicates 
      genetic analysis by fluorescence in situ hybridization (FISH) and polymerase 
      chain reaction (PCR). Fetal hemoglobin, (alpha 2 gamma 2), has been proposed as a 
      useful fetal marker. To improve fetal cell enrichment from maternal blood, we 
      developed an intracellular staining protocol that combines anti-gamma mAb with 
      Hoechst 33342 to identify and flow sort FNRBC. Artificial mixtures of male 
      umbilical cord cells (as a source of fetal hemoglobin) and female adult, 
      non-pregnant peripheral blood mononuclear cells were stained and flow sorted 
      using this protocol. FISH analysis was performed using chromosome X and Y 
      specific probes. Fetal cell purities were calculated by microscope confirmation 
      of anti-gamma staining and counting the number of X and Y signals present after 
      FISH. Results from microscope analyses showed a fetal cell yield of 39-100% and 
      fetal cell purities of 59-73%. These purities are significantly higher than the 
      .001-4.8% previously reported by us in maternal samples using cell surface 
      staining protocols. FISH results demonstrated that 83-100% (mean = 98%) of 
      anti-gamma positive cells were male, whereas 82-100% (mean = 92.5%) of anti-gamma 
      negative cells were female. These results confirmed that the anti-gamma mAb is 
      highly fetal specific. When applied to maternal blood samples, this protocol 
      should lead to increased sensitivity for prenatal diagnosis.
FAU - DeMaria, M A
AU  - DeMaria MA
AD  - Department of Pediatrics, Tufts University School of Medicine, Boston, 
      Massachusetts, USA.
FAU - Zheng, Y L
AU  - Zheng YL
FAU - Zhen, D
AU  - Zhen D
FAU - Weinschenk, N M
AU  - Weinschenk NM
FAU - Vadnais, T J
AU  - Vadnais TJ
FAU - Bianchi, D W
AU  - Bianchi DW
LA  - eng
GR  - P01 HD 18658/HD/NICHD NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Cytometry
JT  - Cytometry
JID - 8102328
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Benzimidazoles)
RN  - 0 (Fluorescent Dyes)
RN  - 9004-22-2 (Globins)
RN  - 9007-49-2 (DNA)
RN  - 9034-63-3 (Fetal Hemoglobin)
RN  - P976261J69 (bisbenzimide ethoxide trihydrochloride)
SB  - IM
MH  - Adult
MH  - Antibodies, Monoclonal/*immunology
MH  - *Benzimidazoles
MH  - Cell Nucleus
MH  - Cell Separation/*methods
MH  - DNA/*blood/isolation & purification
MH  - Erythrocyte Count
MH  - *Erythrocytes/ultrastructure
MH  - Female
MH  - Fetal Blood/*cytology
MH  - Fetal Hemoglobin/*immunology
MH  - Flow Cytometry/*methods
MH  - *Fluorescent Dyes
MH  - Globins/*immunology
MH  - Humans
MH  - *In Situ Hybridization, Fluorescence
MH  - Infant, Newborn
MH  - Intracellular Fluid/chemistry
MH  - Male
MH  - *Maternal-Fetal Exchange
MH  - Pregnancy/*blood
MH  - Prenatal Diagnosis/*methods
MH  - Sensitivity and Specificity
MH  - X Chromosome
MH  - Y Chromosome
EDAT- 1996/09/01 00:00
MHDA- 2000/06/20 09:00
CRDT- 1996/09/01 00:00
PHST- 1996/09/01 00:00 [pubmed]
PHST- 2000/06/20 09:00 [medline]
PHST- 1996/09/01 00:00 [entrez]
AID - 10.1002/(SICI)1097-0320(19960901)25:1<37::AID-CYTO5>3.0.CO;2-B [pii]
AID - 10.1002/(SICI)1097-0320(19960901)25:1<37::AID-CYTO5>3.0.CO;2-B [doi]
PST - ppublish
SO  - Cytometry. 1996 Sep 1;25(1):37-45. doi: 
      10.1002/(SICI)1097-0320(19960901)25:1<37::AID-CYTO5>3.0.CO;2-B.