Replies to comments on Kahang's (Week 7) Post

Hi, I shall answer all your questions here:

To Leslie, Ying Chee & Malerie
I mentioned that 3 different blood smears will be made, one using the patient’s (women’s), one using a male’s and another with cord blood. Remember this test is to test for the presence of fetal cells in the mother’s blood? So the most important factor would be the fetal cells.

Cord blood from newborns is used as a positive control as it definitely contains fetal cells. When stained, they will take up the eosin counterstain and appear as pinkish red intact cells. If red cells are not seen in the positive control, it might be an indication that the stain is not working well.

Male’s blood is used as a negative control because it is impossible that fetal cells will be present. Normal cells of adults will be dissolved after the acid elution staining and appear as ghost cells (illustrations are provided below). This is done to show that the procedures for this test are carried out correctly. If fetal cells are seen in the male’s smear, cross contamination might have occurred or there were errors in the procedures.

In fact the negative control may not necessarily be taken from a male. It can also be taken from a non-pregnant female, as long as it is from an adult. But in certain cases where women are unaware of their pregnancy, the results from the negative control may reflect as positive (presence of fetal cells). So to be more accurate, my lab uses a male’s sample as the negative control.


To Li Ping & Ying Chee
Yes it’s the HbF that affects the resistance to acid elution. The hemaglobin F does not dissolve in the presence of acid and thus takes up the eosin counterstain and appear as red. The adult hemoglobin A cells are already dissolved when acid is introduced, therefore they appear as ‘ghosts’. The reason behind this resistant is not really known. I tried consulting the medtechs in my lab, they are not sure either, just said that it’s definitely related to the HbF component.


To Malerie
The acid elution solution consists of solutions A, B and C:

Solution A – 0.75% Haematoxylin + 96% Ethanol
Solution B – 2.4g of Ferric Chloride + 0.5% Hydrochloric Acid
Solution C – 80% Ethanol

Solutions A, B and C are mixed in the ratio 2:1:1 and this working solution must be prepared fresh.


To Albert, Ying Chee & Gladys
Gladys, yes light microscope would be sufficient. Slides are examined under x40 objective. If fetal cells are observed, counting is performed under oil immersion (x100 objective).

Microscope examination of:

Negative control – Normal adult cell contents are dissolved, therefore nothing can take up the eosin stain, cells appear as ‘ghost’ (cells only have its border with no content)

Positive control (cord blood) – All cells take up the eosin stain and appear as pinkish red intact cells

Positive Result – Stained red cells denote the presence of fetal cells.

(All pictures taken from lab's SOP, with permission from medtech)


Ka Hang
TG02