Foetal RBC

[TDee]

If a maternal sample from a woman who has had a feto-maternal haemorrhage (FMH) is centrifuged where will the foetal red cells lie, at the bottom or the top of the red cell concentrate?

[LaraT23]

Just wondering why you want to know. I mean there are numerous answers depending on the condition and type of the mothers cells and the baby's cells.

The best way to differentiate, I am sure you are aware is by stain or flow cytometry.

[TDee]

This question was raised during a discussion because the commonly held belief is that foetal cells will end up a the bottom of the test tube after centrifugation, however after looking at the BSCH guidelines on FMH, this document states that foetal RBC will sit closest to the plasma/RBC interface. So which is correct? At the bottom or at the top?

[David Saikin]

They should be towards the top of the cell mass.

[LaraT23]

David,

Would you mind explaining that? I know that fetal cells are generally larger and have a higher hemoglobin content, so that automatically makes me think they should be at the bottom. Unless, you are talking like a sed rate type of set up? I just cant get my head around that.

[clmergen]

Centrifugation is based on density not mass (I believe) so fetal cells are less dense and would be at the top (just like retics).

[Eagle Eye]

My thought is same..it will be at the top close to plasma...just like retics.

[RR1]

So, what if the fetal cells are sensitised with maternal antibody... will this affect the density and therefore position after centrifugation?

[Malcolm Needs ☆]

So, what if the fetal cells are sensitised with maternal antibody... will this affect the density and therefore position after centrifugation?

I think this is unlikely, unless, of course, you are using a Svedberg centrifuge.

:D

[RR1]

I think this is unlikely, unless, of course, you are using a Svedberg centrifuge.

:D

I could be using a Svedberg., who knows???, Please explain.

Thanks

xxx

[Malcolm Needs ☆]

I could be using a Svedberg., who knows???, Please explain.

Thanks

xxx

I somehow doubt it!

It is an ultra-centrifuge that spins at VERY high rpm and which was used to discover that IgM was 15S and IgG 7S (the S being the Svedberg).

It takes about a week to stop when it is at full speed.

:D:D

[Malcolm Needs ☆]

I somehow doubt it!

It is an ultra-centrifuge that spins at VERY high rpm and which was used to discover that IgM was 15S and IgG 7S (the S being the Svedberg).

It takes about a week to stop when it is at full speed.

:D:D

Sorry, I mean 19S, not 15S.

I'm a twit.

:redface::redface:

[RR1]

I'm a twit.

:redface::redface:

I don't think so!

[Malcolm Needs ☆]

I don't think so!

Eh? Can I have that in writing?????????????!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

:D:D:D:D

[janet]

Just wondering why you want to know. I mean there are numerous answers depending on the condition and type of the mothers cells and the baby's cells.

The best way to differentiate, I am sure you are aware is by stain or flow cytometry.

Knowing the answer to this has practical use .... some of my staff tend to spin Post Natal Fetal Screens ("we spin all our other samples") and when I tell them that the method states it should be well mixed whole blood they say "oh". I've told them I suspect the fetal cells may centrifuge in a different layer than the adult cells - now I have back up -THANKS!

[TimOz]

I think that a normal bench centrifuge will NOT have any appreciable ability to seperate foetal and adult cells. But a microhaematocrit can do this.

I refer to the AABB Technical Manual method sometimes called "Neocyte seperation". This method uses the same principle of seperating less dense cells from more dense cells. Note that this used a microhaematocrit centrifuge that is running for 15 minutes. These instrument vary a lot but typically run at 6,000 to 15,000 g (RCF) and this seperation is going to be significantly better than a typical bench top tube centrifuge. It is also likely that this method will fail if there is an issue with the density of the 2 populations of red cells so that they have similar densities. I guess that anything that can affect the MCHC will potentially the seperation.

AABB TM Edition 16

"METHOD 2-22. SEPARATING TRANSFUSED FROMAUTOLOGOUS RED CELLS BY SIMPLE CENTRIFUGATION

Principle

Newly formed autologous red cells generally have a lower specific gravity than transfused red cells have and will, therefore, concentrate at the top of the column of red cells when blood is centrifuged in a microhematocrit tube.

This provides a simple method for recovering autologous red cells in a blood sample from recently transfused patients. Note: Red cells from patients with hemoglobin S or spherocytic disorders are not effectively separated by this method (see Method 2-23 for an alternative procedure).

Materials

1. Microhematocrit centrifuge.

2. Plain (not heparinized) glass or plastic hematocrit tubes.

3. Sealant.

Procedure

1. Wash the red cells three times in saline. For the last wash, centrifuge them at 900 to 1000 × g for 5 to 15 minutes. Remove as much of the supernatant fluid as possible without disturbing the buffy coat. Mix thoroughly.

2. Fill 10 microhematocrit tubes to the 60-mm mark with well-mixed washed red cells.

3. Seal the ends of the tubes by heat or with sealant.

4. Centrifuge all tubes in a microhematocrit centrifuge for 15 minutes.

5. Cut the microhematocrit tubes 5 mm below the top of the column of red cells. This 5-mm segment contains the least dense, hence youngest, circulating red cells.

6. Place the cut microhematocrit tubes into larger test tubes (10 or 12 × 75 mm), add saline, and mix well to flush the red cells from the microhematocrit tubes. Then, either 1) centrifuge them at 1000 × g for 1 minute and remove the empty hematocrit tubes or 2) transfer the saline-suspended red cells to a clean test tube.

7. Wash the separated red cells three times in saline before resuspending them to 2% to 5% in saline for testing.

Notes

1. Separation is better if 3 or more days have elapsed since transfusion than if the sample has been obtained shortly after transfusion.

2. The red cells should be mixed continuously while the microhematocrit tubes are being filled.

3. Separation techniques are effective only if the patient is producing normal or above normal numbers of reticulocytes. This method will be ineffective in patients with inadequate reticulocyte production.

4. Some red cell antigens may not be as strongly expressed on reticulocytes as on older cells. Particular attention should be given to determinations of the E, e, c, Fya, Jka, and Ge antigens.

Copyright © 2008 by the AABB. All rights reserved."