Antibody Rule-outs

[bevydawn]

I am just curious as to what rules other facilities follow to do antibody rule-outs. For instance, I worked at one hopsital that required everything be ruled out homozygously except Kell, and C and E when anti-D was present. In those instances we could use three negative heterzygous cells to consider it ruled out. At the hospital that I am currently working, they allow anything and everything to be ruled out if there are 3 negative heterzygous cells and I just do not feel that this is the best practice to follow. Whenever I am performing an antibody ID, I always make sure everything is ruled out homozygously except for Kell (we are a small facility and dont have many panels so homozygous Kell cells are few and far between) and the C and E when anti-D is present. I would like to have this "rule of 3" changed but would first like to see what other facilities are doing.

[donellda]

Our SOP allows for ruleouts to be done on heterozygous cells but I am not comfortable with this either. I usually train new techs to rule out on homozygous wherever and whenever possible especially in the case of Kidds and Duffys. If we cannot find a homozygous cell to rule out then I will use heterozygous cells. Unfortunately, I am not the person responsible for writing SOPs so I just make sure that everyone realizes how important it is to use homozygous cells for ruleouts.

[lef5501]

We are a small hospital as well, and have only one gel panel and one tube panel. However that said, I do make them rule out using at least one homozygous for all clinically significant antibodies except Kell. With an Anti-D, the C and E are usually are hard to find, so they may use heterozygous, but they must have at least three.(which may include the screening cell)

[Mary]

Try saving your expired panels to use for more rule out cells.

[Jane]

We also require everything except Kell be ruled out homozygously. If we can't rule out something then we will give units that are antigen negative for whatever can't be ruled out. A new tech actually missed an antibody last year by ruling out with 3 heterozygous cells. No harm came but it was a good reminder that ruling out heterozygously can get you into trouble.

[jssa1891]

We require the use of homozygous cells to exclude alloantibodies. The Technical Manual supports the use of homozygous cells by advising the use of "cells known to bear a strong expression of the antigen." It also notes that "many antibodies to antigens in the Rh, Duffy, MNS and Kidd systems" show dosage. I have seen a fair number alloantibodies that could have been ruled out using a heterozygous cells so I think it is a risky practice.

[Dawn]

According to the AABB Standards for Blood Banks and Transfusion Services, "When clinically significant antibodies are detected, additional testing should be performed." Well, that is pretty vague...

The AABB Standards for Immunohematology Reference Labs is a little more specific, but leaves a lot to interpretation. "The laboratory shall...Exclude commonly encountered clinically significant antibodies."

Neither of these address zygosity or the need for a certain number of cells for ruling out.

The rules at my facility are:

• D, C, c, E, e, k, Fya, Fyb, Jka, Jkb, S and s must be ruled out with three cells, one of which must be homozygous.
• K must be ruled out with three cells, no homozygous cell required.
• M and N must be ruled out with one homozygous cell.
• Lea and Leb must be ruled out with one cell.
• P1 must be ruled out with one cell, that cell cannot have a weak expression of the antigen.
• In the presence of an Rh antibody, the other Rh antibodies need to be ruled out with three cells, no homozygous cell required. (We do try to find a homozygous cell, but we won't thaw rare cells just to get a homozygous cell.)

Sometimes we have high-frequency antibodies and we can't get the required number of cells for ruling out. In those cases we will get Medical Director approval to deviate from our SOP and go with the AABB Standard.

If we simply can't rule-out we will give antigen negative cells.

[johna]

In my opinion a three-cell requirement for exclusions seems to be quite a bit too much. I've worked in hospitals and reference labs in which a one-cell exclusion, preferably homozygous, was sufficient and I don't recall us ever having any problems with that protocol. With the sensitivity of todays testing methods and reagents I think the chances of missing something by using less than three cells is very remote. I'd find it hard to justify the increased costs of labor and reagents using the protocol you describe.

[OPUS104]

We get workups from our smaller hospitals all the time with only one cell positive. When we work them up in the reference lab we regularly find antibodies that are fairly easily identified eith our more sensitive test. (some only use tube screen) Especially antibodies that only show up with enzyme treatment of some kind or only show up homozygous. This is obviously a subject that should be debated from the stand point of each facility. You could really go overboard here. We have two active bleeders right now with Kpa. Does everyone rule out these odd antibodies? Jsa? Cw? etc.

[Dawn]

We do not have any requirements to rule out anti-Cw, -V, -VS, -Kpa, -Jsa, -Lua. We mostly ignore these. If we suspect that one of these antibodies might be present, then we will pursue an identification.

[sab62]

Hi Ms Debbie Harris

Is it also possible for me to have a copy of your procedure on anitbody ID same with Cybag. Am just a junior staff myself, & we just started doing it. I come from

Phil.

thanks much:)

sab62

my email rfcph@yahoo.com in case you may want to send it.

[ianeke]

We require the use of homozygous cells to exclude alloantibodies. The Technical Manual supports the use of homozygous cells by advising the use of "cells known to bear a strong expression of the antigen." It also notes that "many antibodies to antigens in the Rh, Duffy, MNS and Kidd systems" show dosage. I have seen a fair number alloantibodies that could have been ruled out using a heterozygous cells so I think it is a risky practice.

I agree with this. Our facility is debating the probability issue. How many homozygous cells will confidently rule out an antibody? The 'rule of 3' would be ideal but not always practical. Only using one rule out cell would not allow for the chance that a given cell is not performing correctly and demonstrated a false negative. I have recommended using 'two or more'. Our rule out for Kell is heterozygous, as well as the C/E when anti-D is present (we suggest techs use PEG for these). I dont make the SOP and there is currently a push to reduce the rule out to one cell per antibody. What does AABB recommend? :cool:

[David Saikin]

AABB does not recommend in this situation. Classically, an antibody may be ruled out with one non-reacting homozygous cell, an enzyme pretreated heterozygous cell (when applicable) or when using PeG. These rules have sufficed for years. As I am converting to Gel Technology, I will allow rule outs with heterozygous cells based on the sensitivity of this methodology. Why the uncertainty of the homozygous cell r/o and place yourself in a scenario (occasionally) where you cannot meet your own SOP? But, that's what makes America great!

[ianeke]

AABB does not recommend in this situation. Classically, an antibody may be ruled out with one non-reacting homozygous cell, an enzyme pretreated heterozygous cell (when applicable) or when using PeG. These rules have sufficed for years. As I am converting to Gel Technology, I will allow rule outs with heterozygous cells based on the sensitivity of this methodology. Why the uncertainty of the homozygous cell r/o and place yourself in a scenario (occasionally) where you cannot meet your own SOP? But, that's what makes America great!

The tech manual states, "to lessen the possiblity that chance alone has caused an apparently definitive pattern, there must be a sufficient number of red cell samples that lack and a sufficient number of red cell samples that express, most of the antigens listed in table 19-1." The table refers to Rh,K,Duffy,Kidd,P,Le,MNS. How can we rely on a single example for a rule out?

[Mabel Adams]

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.