Preparing antibodies for students

[ANORRIS]

I seem to have weak reactions when preparing antibodies for students. I am not sure where my problem lies. Can someone share their recipe with me? This sounds absurd, but I can not brew up a good strong batch. (I am making myself sound like a witch...)

[L106]

Of course, the easiest and most true-to-life antibody samples are those from real patients, preferably recent patients. But even if we have to freeze the patient's sample most IgG antibodies seem to work well. But alas....what do you do for patient cells later? Several of us have typed ourselves for various antigens so we might contribute a small sample to serve as the patient's red cells, depending on what antibody(ies) our pt has. (If your antibody pt is Group O, that's great, because you can use an expired panel cell to serve as the patient's red cells.)

If we have to "cook up" a sample, we find that the Rh and K antisera are very potent....just a few drops in a couple milliliters of normal plasma will usually do the trick. We don't usually have much luck with making up samples by adding Kidd, P, Lewis, & Duffy antisera.

Also, you can save old proficiency survey samples for students' use.

Is this the kind of info you were looking for?

[Malcolm Needs ☆]

Of course, the easiest and most true-to-life antibody samples are those from real patients, preferably recent patients. But even if we have to freeze the patient's sample most IgG antibodies seem to work well. But alas....what do you do for patient cells later? Several of us have typed ourselves for various antigens so we might contribute a small sample to serve as the patient's red cells, depending on what antibody(ies) our pt has. (If your antibody pt is Group O, that's great, because you can use an expired panel cell to serve as the patient's red cells.)

If we have to "cook up" a sample, we find that the Rh and K antisera are very potent....just a few drops in a couple milliliters of normal plasma will usually do the trick. We don't usually have much luck with making up samples by adding Kidd, P, Lewis, & Duffy antisera.

Also, you can save old proficiency survey samples for students' use.

Is this the kind of info you were looking for?

If it's any consolation, which I doubt, it is even more difficult in the UK. SInce the problem created by the doctors in Birmingham keeping body parts (including those from babies), we are burdened with the Tissues Act, which means that we have to get written permission from patients if we use any of their samples for anything other than for the reason they were taken (i.e. testing for group, antibodies, cross-match, whatever), and we are breaking the Law if we use the samples for teaching without their express permission.

:mad::mad::mad::mad::mad:

[ANORRIS]

Yes, I was interested in your "cooked up" specimen. Thank you!

[L106]

Malcolm - Thanks for the info. I was not aware of that law in the UK. And how unfortunate....... not allowing the students to work up real patients' antibodies denies them valuable experience. (I know you didn't make the law....I'm just commenting.)

Donna

[Malcolm Needs ☆]

I know you didn't make the law....I'm just commenting.

No, not that powerful...........yet!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

:plotting::plotting:

[babsglab]

I use expired commercial antigens (the ones I use for antigen testing) to make up antibodies for students. Ironically, my concoctions are always too strong! I also make positive DATs by mixing the same antigen typing antiseras with cells testing positive for the sntigen. Those always come out too strong reacting as well!!!!

[AMcCord]

I usually mix 8-12 drops of outdated antisera with 2 mL appropriate type serum or plasma for anti-Fy(a) or (, anti-Jk(a) or (, anti-S and anti-s. It will depend on how strong a particular lot of antisera is - lately my anti-Jks are a bit weaker (12-14 drops seems about right), so you just have to tweak the batch a little sometimes to get the result to want. I start my students out with antibodies that react 2-3+, then as they improve or if I'm feeling evil that day, I cut back on the antisera and give them +/- or 1+ samples.

[webersl]

I use commercial antisera and do doubling dilutions in 6% albumin to find the reaction strength that I am looking for (1+,2+, etc). Then I make up a batch at that concentration. It doesn't always come out right though, and it is time consuming. I do this when I am trying to verify that my staff can accurately grade tube agglutination reactions - it's not really for students. I use real patient serum (frozen) for students. I don't bother trying to match it with red cells. I just teach that concept separately.

[Ajac]

If you really get tight on funds or time. You can always use anti D antisera and manipulate the panels using D+ and D= Rbcs to match the panel anagram. Or use check cells and "regular O cell "suspension for the positive and negatives. (you will have to manipulate screen too, but its cheap and easy)

I found that when making +DAT, be sure to add a small layer of saline on top of cells before adding any plasma or antisera. It seemed to deter the immediate glumping up that sometimes occurs at the juncture point.

:)

[carolyn swickard]

I seem to have weak reactions when preparing antibodies for students. I am not sure where my problem lies. Can someone share their recipe with me? This sounds absurd, but I can not brew up a good strong batch. (I am making myself sound like a witch...)

Be careful with monoclonal source reagents vs. human source reagents. The monoclonal reagents frequently do not react at all like "normal" antibodies. We have some that would not react at AHG phase at all, though they reacted at I.S. as per their normal use instructions. Many of our pt's antibodies are so weak any more we are having real problems coming up with samples for students.

[Dr. Pepper]

Whenever I review panels I look for patients with at least a 1+ antibody and grab all the old lab specs from other depts I can lay my hands on. I pool the serum/plasma and aliquot in 1-2 ml samples and freeze. They seem to keep their reactivity well at -28o. If they're weak you can always do a little demo on differences in sensitivity between LISS, PEG, enzyme, gel etc.

I agree about the monoclonal reagents; if I use them I warn the students to just cross out and look for the specificities and that the human-source ones don't react like that.

Quotient offers free or cheap deals on expired antisera for student use.

[jeanne.wall]

Quotient offers free or cheap deals on expired antisera for student use.

I don’t want to overstep the bounds of information from vendors on BloodBankTalks, but I’d like to clarify the information about Quotient’s support of schools. We have a program that provides in-dated products at low cost for MT/MLT and SBB programs and if we have any products that we are not planning to sell, such as short dated products or trial products, we distribute those free of charge to schools on a first come, first served basis. We also have a Competency Training Kit that provides antibody samples all ready for use.

Hope the information can help someone. Jeanne (Technical Director, Quotient Biodiagnostics)

[mdcbk]

I use the recipes below for students/new techs. We use mostly gel for testing, and these work pretty well most of the time. The Rh, K, and M are very consistent and we get 2+ reactions or better. I don't know what kind of reactions you would get with tube testing with the Rh and K. I've had problems with the Fy, so I only use it in combination with other antibodies. Sometimes I mix it up based on what our expired panels look like. If I'm feeling particularly evil (or want to stump a student who thinks they can solve anything), I will choose several antibodies that are very difficult to identify with a particular panel. We use mostly monoclonal antisera, and I try to use expired, but since I'm only using a drop or two, I don't feel bad about using in-date reagents. I either match the antibodies to an expired panel cell to use as "patient" cells, or use some segments from a phenotyped unit if one is available. I've tried making positive DATs with reagents other than D, but haven't had much success.

• Elution: Anti- D, mixed field DAT
  
  • 2 O Pos Segments and 2 O Neg segments
    
  • Put O pos segments in 12x 75 tube.
    
  • Add 1 drop of Anti-D, 2-3 drops of albumin and a little bit of saline
    
  • Incubate at 37 for 30 minutes
    
  • Add O neg segments
    

  [*]Gel ab screen- Anti- C

  • Fill 12x75 tube 1/3 full of saline
    
  • Add 1 drop Anti-C, 2 drops albumin
    

  [*]Gel ab screen- anti-Jka

  • Fill 12x75 tube 1/3 full of saline
    
  • Add 1 drop Anti-Jka, 2 drops albumin
    

  [*]Gel ab screen- anti-K and Anti- c

  • Fill 12x75 tube 1/3 full of saline
    
  • Add 1 drop Anti-K, 1 drop Anti-c, 2 drops albumin
    

  [*]Tube Ab screen – Anti-M

  • Fill 12x75 tube 2/3 full of saline
    
  • Add 2 drops Anti-M, 2 drops albumin
    

  [*]Gel Ab screen - Anti-Fya, Anti-E, Anti-K

  • Fill 12x75 tube 1/3 full of saline
    
  • Add 2 drops Anti-Fya, 1 drop Anti-E, 1 drop Anti-K, 3 drops albumin