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There are 2 different processes here. The traditional rule of 3 relates to having enough data so that the statistical likelihood of a selected antibody identity being due to chance rather than the real antibody is sufficiently low. Thus, we need at least 3 positive and 3 negative cells to react appropriately (or 2 positive and 5 negative) before we can be sure the reactions are not just due to chance. That has nothing to do with what it takes to rule out an antibody. What we really want to do in rule-outs is avoid giving blood that will cause a frank transfusion reaction. We would also like to avoid giving units that will cause delayed or even "serologic" reactions, if we can. Factors that enter into this are: 1. How strong is the antigen I am testing against? 2. How likely are my reagent cells to be damaged in a way to lose sensitivity? 3. How likely is it that the AHG xm won't catch an incompatible unit? 4. How likely is it that the unit will be positive for the antigen (antigen frequency)? 5. How common is this antibody specificity? 6. Is this antibody specificity known for dosage or anamnestic responses? 7. What kind of reaction is most common if a patient reacts to this antigen (intra- or extra-vascular)? 8. How sensitive is my testing method? 9. How easy is it to find a double-dose cell? 10. How easy is it to find a pos cell in the presence of certain other antibodies? I am sure there are more factors that I have overlooked. What I am describing here is actually Failure Mode Effects Analysis for antibody IDs. Wouldn't that make a great student project? All that said, we don't obsess over finding a double-dose (homozygous) Kpa positive cell to rule it out. We seldom even have a positive cell on our screens. We usually balance the risks (severity of consequences) of missing an antibody against the likelihood of that antibody causing a problem. Anti-K is a common antibody but hard to find a double dose cell to test. The odds of transfused units being K pos are relatively low, so if you miss it on a two unit transfusion, odds are that no more than one unit will be incompatible in vivo (and weakly at that or we would have found it). If you miss an anti-Jka, 75% of units will be incompatible, the reaction is more likely to be intravascular, it's a relatively common antibody, it usually isn't too hard to find a double-dose cell to test against and Kidd antibodies are notorious for being anamnestic. Traditional tube testing panels seem to be pretty robust and seldom show much decrease in antigen strength (as long as no one gets near them with bleach), but pre-diluted gel panels seem like they sometimes lose sensitivity (my observation). But gel is a pretty sensitive method. It is tough to rule out anti-E and anti-C in the presence of anti-D with a double dose cell, but any D negative unit is 99+% likely to be negative for C and E as well, so if you miss it the odds are good you will do no harm. Since none of us likes to think this hard every time we do an ID, we usually create some guidelines that we are comfortable with. I don't see any reason for any more than one double-dose rule-out cell unless you have reason to doubt your panel's integrity. For some specificities and in some multiple antibody cases, a single-dose cell is as good as it is going to get. More than one single-dose cell is only helpful to catch mistakes or if one of the cells has a weaker antigen; testing 2 single-dose cells does not magically make the test as sensitive as testing one double-dose cell. There are many labs that don't even do a repeat ID on a patient with a known antibody unless antigen negative units are crossmatch incompatible and they have not seen any particularly high rate of transfusion reactions. We blood bankers probably worry too much about things that have small impact on the patients. Sorry to rattle on so. I guess I should just write an article for Advance or something.