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Tube method vs Column Agglutination Technology for Grouping


MinerJ
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Hey All,

I have always been told that the use of Tube Method, or conventional tube technique (CTT), is the 'gold standard' for blood grouping and allows a stronger reaction in the reverse group, compared to column agglutination technology (CAT). For a long time I just believed it, since the proof is in the pudding. I have managed to use tube method numerous times to confirm the blood group when it showed weaker expression in the reverse group in the automated CAT. However, I started to wonder why? 

I have looked into it as much as I could but I could not figure out the mechanism behind it, so I was wondering if anyone can shine some light on this?

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Column Agglutination Technology (CAT) is superb at detecting IgG antibodies, and also at detecting certain "cold-reacting" antibodies (such as anti-I and (because of the pH of the column) anti-M, BUT the manufacturers themselves state quite freely, that they are not designed to detect all ABO mis-matches, because most ABO antibodies are IgM (anti-M are usually a mixture of IgM and IgG, but also react preferentially at a low pH).

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Thanks Malcolm

I am going to diverge from my previous question. In regards to Anti-M, you mentioned that these antibodies tend to have an enhanced reaction in lower pH, which is present inside the column. 

In our laboratory, we perform Tube NISS strictly at 37'C to rule out the presence of anti-M reactive at 37'C (mainly for pregnant women). If there is no reaction, then its determined that anti-M is not reactive, and therefore not clinically significant. I was wondering why NISS? Would performing the test using CAT after pre-warming not sufficient? Or is it to do with the enhancement effect of the enzyme? And why NISS over LISS? 

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Hi MinerJ,

We used to do something similar to you when I was in RCI at Tooting, except that we performed tube IAT strictly at 37oC, with warmed NISS for the washing process.  As far as this is concerned, I could never see any difference between having the red cells initially suspended in NISS or LISS, except, of course, that the incubation time was shorter with LISS (although we used to bring the reactants to 37oC before the initial mixing).  Again, like you, this was mainly for pregnant women.

After many experiments trying to get the CAT reaction chamber strictly up to 37oC BEFORE the addition of the reactants, and failing miserably, we abandoned the idea, as we could never quite get to 37oC.  Unfortunately, "cold reacting" antibodies can (and do) sensitise their cognate antigens extremely quickly, but are much slower to dissociate, so we were never quite sure whether the anti-M was reacting genuinely at 37oC or not.

I think you may have made a typo.  There are no enzymes involved, because of course, the M antigen is papain sensitive, by the columns have a slightly low pH, and this, together with centrifugation and the difficulty in keeping the reaction chambers at strictly 37oC all mitigate towards the possibility of a false positive at 37oC.

I hope this bit of rambling answers your question sufficiently, but, if not, do not hesitate to get back to me (or anyone else, come to that!).

Malcolm

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