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While the use of EDTA plasma basically eliminates the chance of detecting hemolytic antibodies (as Malcolm says above), some laboratories still use serum. It's possible some hemolysis might been seen in visual check before taking the serum-RBC-PEG reactants to IAT. One may also recognize loss of RBC volume after the washing process, if some of the cells were destroyed during the incubation phase.

1. Personally, I wouldn't do ANYTHING different/extra than the manufacturer recommends. You may inadvertently "modify" the process and find yourself in a corner that requires validation of your local modification. Yikes ! 2. Typical eluate volumes are quite small - often too small to be measured with a pH probe. So I suspect, correct me if I'm off-base, that the pH has been checked using pH paper. If so, eyeballing the color of the pH paper is no better than eyeballing the color of the eluate. And, as AMcCord suggests, while the blue color may vary, the differences in pH values of the different blues is very small. The manufacturer (Gamma/Immucor) has put a lot of effort into making the kit idiot-proof.

The pH indicator used in the Buffering Solution would be specific for the desired pH range. If you see a blue color, the solution is in that fairly narrow range - chemistry at work. There is no requirement in the insert (or in standards that I'm aware of) to check the pH by a method other than eyeballing the color.

The Immucor package insert for PeG says that tubes 'may' be examined after the 37C incubation for hemolysis w/o centrifugation.

And to you too MyHerpesItch. Well, yes and no! EDTA certainly does chelate Ca++, but it also chelates Mn++ and Mg++, and all three are required as co-factors for the Classical Complement Pathway, rather than actually being part of complement, which is why you don't see immune-mediated haemolysis.

We never did, as far as I can remember.

You can use either. For products with an expiration of less than or equal to 72 hours, you must use the exact time. For products with an expiration of more than 72 hours, you can use the number of days @2359 (Technical Manual, 16th ed, pg 219). We have been using the 120 hrs but are in the process of changing to the 5 days @2359.