A consultation about antibody clinical significant testing

[Yanxia]

I want to test whether an antibody is clinical significant , but I cann't use Cr to label the red cells. Would some friends kindly give me some advice about what I can use to know whether the cells is destroyed by the antibody? Can I use LDH , bilirubin and free hemoglobin?

Thank you for your helping!

[Yanxia]

I don't know if I can use flow cytometry to do it. The question is I can get the survival time of red cells but I can't know whether the cells is destroyed or not, because maybe some cells with less antigen can survive normally, does it possible? Or I can testing survival time in parellel in a patient and a healthy person.

I don't know how to do the testing. Would someone give me any suggestion? Thank you!

[David Saikin]

You might want to try setting up a monocyte monolayer assay . . . you can see if the patient's monocytes will "attack" red cells sensitized with the antibody. Someone else will have to help you with the particulars, I have only read about this assay.

[Yanxia]

Thank you, David.

It is a good method, but I can't do it in our hospital. Such a pity!

[Mabel Adams]

To use flow cytometry you would need to have a system that could destroy the cells like they would be in vivo if you transfused them. If the antibody is a complement fixer, this might work in vitro, but if you need an intact immune system (preferably the intended recipients's) to destroy the cells if the antibody is significant, I can't think of any way to make that work in the lab except maybe the monocyte monolayer assay. Is there any way it could be adapted to flow cytometry? I am speculating heavily and probably don't know what I am talking about.

There is a method called the "in vivo crossmatch" where you give the patient a small amount of the unit (like 20 cc) over 15 minutes then draw a sample from the patient and look for visible hemolysis (or measure free hemoglobin). If no overt signs of destruction are present, the unit is considered compatible, but this is a very insensitive method and could be risky to the patient.

You could look for bilirubin, but bilirubin peaks about 6 hours post-transfusion so that wouldn't be very helpful. I would think LDH would parallel hemolysis, but you would need to know the patient's pre-transfusion level and I wouldn't trust it if the pre- were abnormal maybe due to underlying organ damage.

[Yanxia]

Thank you, Mabel!

I want to transfuse the cells to the patient's body, then test the survival of the cells. Because the transfused cells' type is different, I can use flow cytometry to test the quantity of it and draw the survival curve. With antigen negative cells as parellel.

The antibody's clinical significance is unknown, some blood bank don't choose negative cell give the patient. It a bit like anti-D with Del cells.

[Yanxia]

The patient with anti-D we will give them D negative cells, but those cells maybe Del positive. I do this testing to detect whether this kind of case can reduce the cells life time. This comparison is not exact, just a little like this.

[Mabel Adams]

Is the antibody you are testing something with a known specificity or a new one, never before identified? I am curious what antibody you are working with.

[Yanxia]

I want to know whether A&B subgroup antibody is clinical significant. But to transfuse a patient with room temperature reactive antibody is so dangerous, maybe I will do retrospective research first. I do this test because I have not read any reports of HDFN induced by A&B subgroup antibody.

[Mabel Adams]

As I recall, many babies will type as A subgroups even if they will be A1 as adults because the ABO antigens are not completely developed at birth. At least we had to wait till my daughter was a few months old before testing to see if she was an A2 like her dad or an A int like me (no, we didn't poke her just to find this out, and she is A2).

For it to cause HDFN, it would have to be IgG so it can cross the placenta. Do you have examples of anti-A1 that are IgG?

[Yanxia]

Thank you, ,Mabel.

I have not saw any A&B anti-subgroup antibodies which is IgG. If a mother is A2 or Ax have naturally produced anti-A1 , during her pregnancy the baby's cellscan enter her blood circulation stimulate the B cell to produce antibodies. As we have not saw any this kind of IgG antibodies in subgroup people. Would I get the conclusion that A&B subgroup immunology can't chang the antibodies to IgG, they have not the ability of evoke B memory cells?

In China I have not saw any reports of warm reactive subgroup antibodies, not like U.S., this maybe the difference between human race, So I am very interesting in this antibodies' investigation.