Enzyme Panels & The DAT

• alternative methods used to enhance antibody activity or to decrease its sensitivity: PEG (polyethylene glycol), LISS; Enzymes; increased incubation time; increased serum to cell ratio
• the most commonly used enzymes are ficin and papain
• ficin is from figs
• papain is from papaya
• Ficin and papain destroy or weaken antigens such as M, N, Fya, Fyb, Xga
• ficin and papain enhance the activity of Rh, I, P, Kidd, and Lewis
• some antibodies react better at RT or below, including anti-M, N, P1, Lea, Leb, A1
• inhibition technique is using something to get rid of a particular antibody
• inhibition technique

  e.g. if you want to confirm that you have anti-P1, you can add pigeon egg fluid to one sample and saline to another and run against a panel with P1 positive cells

  if you suspected anti-P1 of blocking another antibody, you could absorb out the anti-P1 with pigeon egg fluid and then rerun the panel
• P1 - hydatid cyst fluid or pigeon egg whites
• Sda - human urine (from a Sd(a+) individual)
• chido, rodgers - plasma from Ch+, Rg+ individuals
• adsorption technique

  • adsorption is the removal of an antibody from a serums ample onto red cells that express the corresponding antigen
• adsorption

  • after separation of serum and red cells, the specific antibody remains attached to the red cells
  • one can harvest the antibody by elution and test for it or examine the adsorbed serum for antibodies remaining after the adsorption process
• e.g. adsorption

  • possible anti-e and anti-S but not sure
  • take pt serum, and e+, S- red cells
  • incubate together
  • spin and take away supernatant, run against panel
  • now able to detect anti-S
• Adsorption technique is useful when there are multiple antibodies present in a single serum, removing autoantibody to permit ID of underlying antibodies
• multiple antibodies may be suggested if

  • panel reactivity doesn't fit pattern of exclusion
  • reactivity is present at different test phases
  • unexpected reactions obtained when attempts are made to confirm specificity of suspected Ab
  • no discernable pattern
• multiple antibodies

  • we phenotype patients in order to ascertain their antigenic makeup and narrow down an antibody specificity; keep in mind the patient will not have an antibody to an antigen they possess; if they did have an autoantibody, the auto control would be positive
• high prevalence antigens

  • antibodies to a high prevalence antigen
  • when the auto control is nonreactive, but all the panel cells are positive, an alloantibody to a high prevalence antigen should be expected
• high prevalence antigens

  • usually need a reference lab for workup
• Knopps (Kna) - 100% in black population, 99% in Caucasian population

  would need to send the sample to a reference lab that actually has cells negative for Kna in order to find another possible second antibody

  or would need to have anti-Kna antibody serum to test the patient cells which would be negative for this high prevalence antigen
• low prevalence antigens

  • antibodies to low prevalence antigens
  • serum usually reacts with a single donor or reagent cells sample
  • most low prevalence antibodies are reactive at temperatures below 37 degrees C and therefore have doubtful clinical significance
• low prevalence antigens

  • may be able to find a single panel cell with the antigen
  • sometimes we find this whena. crossmatch is positive, even though the screen was negative; in this case patient has antibody to antigen not present in panel cells, but donor happens to have that antigen
• selecting blood for transfusion

  • after an antibody has been identified, it's clinical significance must be determined
• selecting blood for transfusion

  • whenever possible, all units of red cells selected for transfusion to a patient with a clinically significant antibody should be antigen-typed and must be negative for the corresponding antigen
• selecting blood for transfusion

  • even if the antibody is no longer detectable, all subsequent RBC transfusions to that patient should lack the antigen in order to prevent a secondary immune response
• rare blood includes units that are negative for high-prevalence antigens, as well as units that are negative for a combination of common antigens
• when a patient has multiple antibodies, we need to determine to prevalence of compatible donors
• you determine the prevalence by multiplying the prevalence of donors negative for each of the antigens

  if you need a certain number of units, divide what you need by this number
• auto control and the DAT

  • the autologous control tests the patient serum and autologous red cells that are tested under the same conditions as the serum and reagent cells
• A DAT is done if the autocontrol is positive
• DAT is direct antiglobulin test; it is done to see what is coating RBCs; it is in vivo testing (coats red cells IN patient)

  • done by mixing washed cells with AHG and look for agglutination
• DAT is also done when HDFN is suspected, when there is a transfusion reaction, and to work up autoantibodies
• some healthy individuals have positive DATs, and some patients with negative DATs have immune hemolytic anemia
• negative DAT

  • if the DAT is negative, sometimes the amount of IgG coating the RBCs is lower than can be picked up with commercial IgG, may be sent out
  • IgM or IgA antibodies may be on the RBCs but are not picked up by anti-IgG (AHG)
  • e.g. cold agglutinins can be IgM (used prewarmed technique to transfuse)
• positive DAT

  • autoantibodies can cause WAIHA
  • previous transfusions - antibodies are made that coat donor cells before we see antibodies in the serum
• WAIHA - may be better not to transfuse, as recipient will attack donor cells, causing increased, bilirubin, etc
• We can see recipient antibodies coating donor cells in 7-10 days after a transfusion (1-2 if it is an anamnestic response). we won't see antibodies in the serum for 14-21 days because they coat the RBCs first and then the excess is found in serum
• positive DAT

  • drugs - penicillin in high doses can bind to RBCs in vivo; if patient has antibodies to penicillin, they will absorb onto the drug
  • if we did an elution, it would be negative, as the antibodies are binding to penicillin, not the RBC antigens; if we eluted off and rand the antibodies against a panel, it would be negative
• Investigating DAT+

  • we can perform an elution - it separates the antibodies from the RBCs
  • it works by changing the thermodynamics of antigen-antibody reaction
  • it does this by neutralizing (or reversing) forces of attraction that hold antigen-antibody complexes together or by disturbing the structure of the antigen-antibody bindings ite
  • the idea is to recover the bound antibody in a usable form