Making ABO discrepancies for teaching

[jbeauvais]

I'm having trouble making examples of Anti-A1 for teaching purposes. I've been making them for years using A2 patients with A1 lectin & pt. serum. Lately it seems that when I make and test the sample it's 4+ right after I make them. The next morning the reactions are negative. Anyone have a recipe for making and keeping an Anti-a1 reactive?

Jeanne

[donellda]

Hi Jeanne! It's me, Donna, from Sinai Grace. Welcome to Bloodbanktalk! I have the same problem. The only thing that works for me is to add the Anti-A1 early in the morning when the students are going to work on the unknowns. Maybe someone else has a good secret for us.

[jbeauvais]

I guess I should have tried to be a little more creative with the logon. Can't hide anywhere.

[Mabel Adams]

On another student mock sample need: does anyone have a good recipe for faking weak D blood?

[rcurrie]

Sure Mabel! But don't stop with weak D- toss in the one that is DAT pos so they will recognize the difference.

Weak D: 1 drop R2R2 + 19 drops rr

Pos DAT: 2 drops CCC + 18 drops rr

My students come away knowing the difference between a weak D and a positive DAT.

As for the A2 with anti-A1, the A1 lectin does indeed deteriorate quite quickly. When I make it, I hand it to the student and say, "do an ABO/Rh on this one RIGHT NOW."

BC

[kelliott]

The recipe for weak D mentioned above sounds more like a good sample for a mixed field reaction. If you use that for weak D - how then will you teach them the difference between weak D and a mixed field reaction?

For the A2 with anti-A1 - try using an A2 that is a nonsecretor.

Ask your blood supplier - or reference lab to send you some examples of weak D. That has worked for us.

A good recipe for a weakly positive DAT is to take 10 mL of a 5% red cell suspension of D positive cells. Add 1-2 drops of human anti-D, incubate for 30 minutes at 37C, wash 5x and resuspend to 3-5%.

[rcurrie]

Yes, it kind of looks like MF. But, the whole idea is to get the student to question the weak D by performing a DAT whenever they get a suspected weak D. I am my blood supplier and reference lab, and we just don't find weak D donors that often.

A much simpler weak positive DAT is to simply dilute some Coombs check cells, and that is what I do.

BC

[Yanxia]

I have an interesting qustion: Is every A2or A2B person with anti-A1 nonsecretor?

[Mabel Adams]

Bob, whose CCC do you use? Mine are apparently at least partly Rh pos so don't work well in your recipe--it comes up weak pos to 1+ at IS with anti-D.

The other recipe is, as we would expect, rather more mixed field, but workable. Thanks.

My supplier sends out their donor testing so doesn't have many weak D samples either.

[kelliott]

Unless things have changed dramatically coombs control cells are all D + coated with anti-D.

secretor versus non secretor - individuals of the Le(a+b-) phenotype are non secretors.

[Yanxia]

secretor versus non secretor - individuals of the Le(a+b-) phenotype are non secretors.

And part of Le(a-b-) are non secretors.

[rcurrie]

Mabel, we use Ortho Coombs Control (Weak). You may have to play with

the formula a little. Also, if the student incubates the cells with

anti-D for any period they may see a 1+w. I tell them up front that

they are looking at a simulation, and that it is not perfect. The whole

idea with my exercise is to get the student to question a "weak D" by

doing a DAT. I teach them to know their patients (pregnant, recently

transfused, etc.).

BC

[rcurrie]

I finally had a true weak D donor yesterday. I am going to flag this donor and hope he donates on a regular basis. I was able to keep some segments from his current donation so that I will have weak D samples for my 2007 students.

BC

[marilynm]

If you can find a group A2B from your employees, a blood supplier's donors, etc, then you could just use anti-A1 lectin without adding it to anything. I know A2B samples might be hard to find, but if you do find one, you can keep it for a long time if you store the cells in a preservative solution. mmoulds

[Eagle Eye]

Hi, Maryline,

Wow! We have expert in Immunohematology posting comments on this forum.

Can you share the procedure on saving those cells for longtime? If you do not want to post here can you please e-mail it to me?

Thank you in advance.

[rcurrie]

I usually have about 20 AB units on my shelves. I will screen them for A2 and add some Alsevers solution to some cells from segments. Thanks for the great idea, Marilyn!

BC

[Mabel Adams]

Marilyn, I heard you had retired. Glad to see you aren't just tending roses or touring Europe but have stayed to keep all of us blood bankers educated.

[Yanxia]

Hello,Marilyn.

I come from China.I come here to learn bloodtransfusion medicine and English.I am glad to see you serologist at here.

I have a question,if you have time,please help me:why anti-A1 lectin added to A2 person's serum will become weaker . Can use a nonsecretor's serum resolve this question?

Thank you!

[rcurrie]

Shily, my guess is that it is a dilutional effect. A1 lectin will agglutinate all type A cells until it is properly diluted to only agglutinate A1 cells.

BC

[marilynm]

Commercial Anti-A1 lectin has been diluted so that it will only react with A1 cells. Adding it to human plasma will definitely dilute it, but it is pretty strong in reactivity so it should be able to be diluted quite a bit. I think the question is "why it gets weaker after a day or so after being added to human plasma?". when you mix monoclonal antibodies with human antibodies of the same specificity (Rh D for instance), you sometimes get a weakening of the human antibody. Perhaps the human ABO isoagglutinins are affecting the Anti-A1 lectin (prepared from seeds). I will ask my manufacturing experts and get back to you! Marilyn PS I too have to consult experts for some questions! Sorry to be so longwinded, which is my character, as you know. You may be sorry that I am back!

[Yanxia]

Thanks Rcurrie and Marilynm.

I guess mybe because the A substance in the A2 plasma reacted with the lectin.

Marilynm says "when you mix monoclonal antibodies with human antibodies of the same specificity (Rh D for instance), you sometimes get a weakening of the human antibody"

I am very interesting in it. May I trouble you to explain it ?

[Mabel Adams]

Shily, your question about the A substance is interesting. Marilyn, is it possible that the A substance in the serum used to dilute the anti-A1 lectin could adsorb out the lectin in addition to the other cause of weakening that you mentioned?

Maybe I could do some experimenting because I am married to one A2 that is Lea and Leb neg and I gave birth to another. I am A int and Leb pos so my daughter could be a secretor. I am not sure I have time or materials to do secretor studies on them, but I could probably provide someone some samples.

I could do the A1 lectin dilution with my husband's A2 (possible non-secretor) serum/plasma and see if reactivity drops off like it does for everyone else.

[Yanxia]

Mabel Adams, I want to say you are so lucky. Your husband is Le(a-b-), perhaps he is a secretor, there is a little trouble. And Le(a-b-) secrete person will have more ABH substance in their plasma/serum than other person. I think test his saliva substance before do dilution is better.

[Mabel Adams]

OK, I am reading the secretor procedure in the Tech Manual. I don't have anti-Lea nor anti-H lectin, but I suppose we don't really care about anything but A substance for this experiment. My only source of polyclonal anti-A would be a patient or volunteer. I can come up with a positive control secretor, but not sure I could come up with a negative control. The other problem I have is time. Does anyone have any of that to loan me?

[Yanxia]

Mabel Adams, I have several questions:why we can't use monoclonal anti-A in this test? And I don't understand the meaning of a positive control . I think you can use normal saline instead of anti-A as negative control.