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Epsilon haemoglobin specific antibodies with applications in noninvasive prenatal diagnosis.

Sørensen MD, Gonzalez Dosal R, Jensen KB, Christensen B, Kølvraa S, Jensen UB, Kristensen P - J. Biomed. Biotechnol. (2009)

Bottom Line: Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial.DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry.Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Aarhus, Denmark.

ABSTRACT
Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising epsilon-Hb, one was chosen for further characterization, DAb1. DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry. Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of the method, K562 cells (which express epsilon-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis.

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Staining of enriched maternal blood sample taken as post-CVS. First enrichment performed by CD-71 directed MACS. Subsequently cells were mounted on glass slides and permeabilized with methanol followed by immunostaining with FITC conjugated DAb1 (30 μg/mL). Subsequently FISH was performed to karyotype the cells. Embryonic nucleated red blood cells (primitive erythroblasts) identified with DAb1, directly conjugated with FITC (green) is shown. Subsequently, FISH was performed against both the X- (blue) and Y-chromosomes (red), confirming the origin of the immunostained cells. X-chromosomes of nonimmunostained nucleated maternal cells (seen as blue dots), are located around the identified foetal erythroblasts.
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fig2: Staining of enriched maternal blood sample taken as post-CVS. First enrichment performed by CD-71 directed MACS. Subsequently cells were mounted on glass slides and permeabilized with methanol followed by immunostaining with FITC conjugated DAb1 (30 μg/mL). Subsequently FISH was performed to karyotype the cells. Embryonic nucleated red blood cells (primitive erythroblasts) identified with DAb1, directly conjugated with FITC (green) is shown. Subsequently, FISH was performed against both the X- (blue) and Y-chromosomes (red), confirming the origin of the immunostained cells. X-chromosomes of nonimmunostained nucleated maternal cells (seen as blue dots), are located around the identified foetal erythroblasts.

Mentions: To establish the optimal permeabilisation procedure for cell immunostaining, the antibody fragments were tested in Dot-blots where cells treated with different permabilisation regimes were tested (data not shown). The optimal treatments of the cells were with methanol alone or mixed with acetone (1 : 1 or 1 : 3), followed by storage at −20°C over night. Initially immunocytochemistry with antibody DAb1 performed on methanol fixed and permeabilised K562 culture cells showed intense staining, compared to nonstained cells (data not shown). One of the antibodies gave a significant better staining than the other three antibodies, the DAb1. DAb1 stained approximately 10% of the cells in an embryonic blood sample (first trimester) demonstrating the presence of several ε-Hb containing erythroblasts (data not shown). A blood sample taken post-CVS (week 10 + 3) from a woman carrying a male foetus was enriched for foetal cells by CD-71 directed MACS and subsequently analysed as well. MACS-enriched cells obtained from a 2 mL blood sample were coated on slides. The two slides were permeabilised with methanol, and immunostaining was subsequently performed with DAb1. After confirming that the slides contained stained cells, FISH was performed against the X- and Y-chromosomes. Scanning of the slides revealed 129 cells identified as foetal erythroblasts, since they were positive by both Y-chromosomes directed FISH and immunocytochemistry for ε-Hb (Table 2 and Figure 2). This corresponds to one embryonic NRBC pr 7.8 × 107 maternal blood cells. Several cells were damaged during the FISH procedure because methanol is not strong enough as a fixative for the harsh conditions associated with FISH analysis. Subsequent staining, after paraformaldehyde fixation, revealed as strong or stronger staining of cells (not shown), and makes the cell more resistant towards the FISH procedure, which will lead to a higher FISH efficiency.


Epsilon haemoglobin specific antibodies with applications in noninvasive prenatal diagnosis.

Sørensen MD, Gonzalez Dosal R, Jensen KB, Christensen B, Kølvraa S, Jensen UB, Kristensen P - J. Biomed. Biotechnol. (2009)

Staining of enriched maternal blood sample taken as post-CVS. First enrichment performed by CD-71 directed MACS. Subsequently cells were mounted on glass slides and permeabilized with methanol followed by immunostaining with FITC conjugated DAb1 (30 μg/mL). Subsequently FISH was performed to karyotype the cells. Embryonic nucleated red blood cells (primitive erythroblasts) identified with DAb1, directly conjugated with FITC (green) is shown. Subsequently, FISH was performed against both the X- (blue) and Y-chromosomes (red), confirming the origin of the immunostained cells. X-chromosomes of nonimmunostained nucleated maternal cells (seen as blue dots), are located around the identified foetal erythroblasts.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712645&req=5

fig2: Staining of enriched maternal blood sample taken as post-CVS. First enrichment performed by CD-71 directed MACS. Subsequently cells were mounted on glass slides and permeabilized with methanol followed by immunostaining with FITC conjugated DAb1 (30 μg/mL). Subsequently FISH was performed to karyotype the cells. Embryonic nucleated red blood cells (primitive erythroblasts) identified with DAb1, directly conjugated with FITC (green) is shown. Subsequently, FISH was performed against both the X- (blue) and Y-chromosomes (red), confirming the origin of the immunostained cells. X-chromosomes of nonimmunostained nucleated maternal cells (seen as blue dots), are located around the identified foetal erythroblasts.
Mentions: To establish the optimal permeabilisation procedure for cell immunostaining, the antibody fragments were tested in Dot-blots where cells treated with different permabilisation regimes were tested (data not shown). The optimal treatments of the cells were with methanol alone or mixed with acetone (1 : 1 or 1 : 3), followed by storage at −20°C over night. Initially immunocytochemistry with antibody DAb1 performed on methanol fixed and permeabilised K562 culture cells showed intense staining, compared to nonstained cells (data not shown). One of the antibodies gave a significant better staining than the other three antibodies, the DAb1. DAb1 stained approximately 10% of the cells in an embryonic blood sample (first trimester) demonstrating the presence of several ε-Hb containing erythroblasts (data not shown). A blood sample taken post-CVS (week 10 + 3) from a woman carrying a male foetus was enriched for foetal cells by CD-71 directed MACS and subsequently analysed as well. MACS-enriched cells obtained from a 2 mL blood sample were coated on slides. The two slides were permeabilised with methanol, and immunostaining was subsequently performed with DAb1. After confirming that the slides contained stained cells, FISH was performed against the X- and Y-chromosomes. Scanning of the slides revealed 129 cells identified as foetal erythroblasts, since they were positive by both Y-chromosomes directed FISH and immunocytochemistry for ε-Hb (Table 2 and Figure 2). This corresponds to one embryonic NRBC pr 7.8 × 107 maternal blood cells. Several cells were damaged during the FISH procedure because methanol is not strong enough as a fixative for the harsh conditions associated with FISH analysis. Subsequent staining, after paraformaldehyde fixation, revealed as strong or stronger staining of cells (not shown), and makes the cell more resistant towards the FISH procedure, which will lead to a higher FISH efficiency.

Bottom Line: Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial.DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry.Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Aarhus, Denmark.

ABSTRACT
Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising epsilon-Hb, one was chosen for further characterization, DAb1. DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry. Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of the method, K562 cells (which express epsilon-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis.

Show MeSH
Related in: MedlinePlus