IgM vs IgG

[LAURAA80]

We have solid phase technology and have been missing antibodies. We were re-validating our instrument due to missed antibodies. We test at the bench using PeG as our reference method. We have been picking up antibodies at the bench (with IgG only, not at immediate spin) and when testing with solid phase we do not pick up weak antibodies. Samples sent to the company for determining the discrepant results are always determined by the company to be IgM and solid phase does not pick up IgM. We don't see reactivity at immediate spin and use IgG. Is there a way to determine IgM antibodies that react only at coombs phase? Does IgG really pick up IgM antibodies frequently? Aren't IgM antibodies generally associated with complement? Any help with this would be appreciated.

[David Saikin]

Are the abs you are missing (IgMs) clinically significant? One of the reasons to use anti-IgG is to NOT find these clinically insignificant, cold abs. Anti-IgG should not find IgM abs, but - occasionally these abs will react strongly enough so that their agglutination will carry throught to AHG phase.

[JEMarti]

It's hard to say what's happening from the information provided. You say that you are finding Ab's that the solidphase is missing when you repeat testing on the bench and that the reactions re weak at AHG phase. Your solid phase manufacturer claims that the Ab's are IgM.

Since you use PEG you could be finding IgM antibodies that are weak as they do not have enough time to react at IS, then when you add PEG and incubate you do not read at 37 prior to washing and adding AHG. The reactions you see are possibly IgM that is weak and it has a broad enough thermal range to still be active at 37 and just needs enough incubation time to become evident.

You didn't say what Ab's you are seeing or whether you have been able to make ID's on them. That information would be helpful.

[LAURAA80]

Thank you both for the information. We are able to pick up these antibodies at the bench in PeG. The antibodies automation has missed are Anti-E, K and Jka....and there were at least two of each. In each example there was no reactivity seen at immediate spin, but was clear cut at AHG. Most of these were newly identified antibodies, which could be IgM, but I am still unsure how to differentiate given the situation. When validation a new piece of equipment, I would like to know for sure antibody is IgM so when solid phase or gel reactions are negative, I don't consider reactions as false negatives. Please let me know if you have any additional insight.

[JEMarti]

If you wanted to differentiate IgG and IgM antibodies you could DTT treat the plasma to get rid of the IgM and then you would only see the reactions from IgG.

We saw the same issue when we went to solid phase. We saw a number of antibodies that did not react in solid phase or in the tube with LISS but they did react in PEG. We concluded that it seemed to be more an issue of the LISS rather than solid phase since the solid phase is a LISS technique. I think that ultimately you need to make your peace with the fact that every medium has it's faults and unless we want to screen using multiple media we will always run the risk of missing something.

[Malcolm Needs ☆]

If you wanted to differentiate IgG and IgM antibodies you could DTT treat the plasma to get rid of the IgM and then you would only see the reactions from IgG.

We saw the same issue when we went to solid phase. We saw a number of antibodies that did not react in solid phase or in the tube with LISS but they did react in PEG. We concluded that it seemed to be more an issue of the LISS rather than solid phase since the solid phase is a LISS technique. I think that ultimately you need to make your peace with the fact that every medium has it's faults and unless we want to screen using multiple media we will always run the risk of missing something.

Totally agree with both your post and David's.

:):)

[adiescast]

We actually did not see any missed antibodies when we went with solid phase (about 12 years ago), but we went from LISS tube method. We actually detected more antibodies by solid phase than by LISS tube method. We have recently seen that manual reading of the solid phase plates will detect more antibodies than automated reading of the plates. That is causing some controversy in our lab.