Development of a preparation and staining method for fetal erythroblasts in maternal blood: Simultaneous immunocytochemical staining and FISH analysis

Development of a preparation and staining method for fetal erythroblasts in maternal blood: Simultaneous immunocytochemical staining and FISH analysis

In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable staining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slides. Chorionic villi w...

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Published in:Cytometry (New York, N.Y.) Vol. 32; no. 3; pp. 170 - 177
Main Authors: Oosterwijk, Jan C., Mesker, Wilma E., Ouwerkerk‐van Velzen, Maria C. M., Knepflé, Cecile F. H. M., Wiesmeijer, Karien C., van den Burg, Marja J. M., Beverstock, Geoffrey C., Bernini, Luigi F., van Ommen, Gert‐Jan B., Kanhai, Humphrey H. H., Tanke, Hans J.
Format: Journal Article
Language:English
Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-07-1998
Subjects:
3,3'-Diaminobenzidine
Centrifugation, Density Gradient
diaminobenzidin
erythroblast
Erythroblasts - chemistry
Erythrocyte Count
Female
fetal cells in maternal blood
fetal hemoglobin
Fetus - chemistry
Fetus - cytology
Ficoll
FISH
Humans
immunocytochemistry
Immunohistochemistry
In Situ Hybridization, Fluorescence
Male
Pregnancy
prenatal diagnosis
Staining and Labeling
Tissue Fixation
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Summary:In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable staining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slides. Chorionic villi washings, cord blood, and maternal blood samples were used for this study. After a density gradient separation and centrifugal cytology, slides were stained either with 3,3‐diaminobenzidin (DAB), a marker for heme, or with antibodies against the γ‐chain of fetal hemoglobin (HbF). FISH analysis for both X‐ and Y‐chromosomes was performed on the same slides. Cytocentrifugation provided a controlled cell density on the slides with good cell morphology. Both the DAB and HbF staining were suitable for manual screening of large numbers of slides. The HbF staining, although supposed to be more specific for fetal NRBCs, appeared to be more sensitive to minor changes in preparation. We were eventually able to combine HbF staining with FISH analysis, and produced a detection efficiency of >85% for both X‐ and Y‐chromosome signals. This preparation protocol simplifies the detection of NRBCs in maternal blood. Immunocytochemical staining and FISH analysis can be performed on the same cell with good image contrast, thus facilitating both manual and automated image analysis. This will facilitate the use of this approach for prenatal diagnosis. Cytometry 32:170–177, 1998. © 1998 Wiley‐Liss, Inc.
Bibliography:Preliminary results of this study were presented at the 18th Congress of the International Society for Analytical Cytology in Rimini, Italy, April, 1996.
ObjectType-Article-1
SourceType-Scholarly Journals-1
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content type line 23
ISSN:0196-4763
1097-0320
DOI:10.1002/(SICI)1097-0320(19980701)32:3<170::AID-CYTO2>3.0.CO;2-O