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Still upgrading SOPs on rule in/rule out antibodies


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HI all and my apologies if I've asked this before in one post or another, I just can't keep track these days. Anyway, we have a poor, unclear procedure here for ruling in and ruling out antibodies when reading antigrams. I should mention that we don't do anything beyond routine panels, no  enzyme panels or other fancy stuff. We are a tube method, very small volume,  lab but within a couple of weeks should be solid state (Echo) users. :D :D :D Might as well get a decent process in place now that is applicable to both technologies when cells in the panels are positive. I should mention that most of our techs are crosstrained and can go weeks before working in the Blood Bank. So they all need a decent procedure to have as a reference.

 

Currently, we rule out antibodies based on the first negative homozygous cell we get for a specific antibody in a 10 panel screen (manual, I think the Echo has many more cells for the Echo). One negative cell that should be positive, for example, for anti-C , and out it goes!  I don't think that's enough and I know at least the wonderful Malcolm has said this also.

 

Ruling IN is another story. We don't actually tell our techs how many positive cells they need to find to rule IN an antibody that may be overlapping another one. For example, we had a clear anti- Fya the other day where the same  cells were positive for anti-C. The tech found one Fya neg cell that was homozygously positive for anti C, got a positive result and declared that the patient had anti-C. But another tech tested a couple of other Duffy neg cells that were homozygous pos for anti C and they were negative.

We need clarity! How many cells do you use to rule out an antibody and how many to rule in an antibody. This is a poll. Thanks for your help and Happy Weekend!!

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The 3 and 3 rule is fairly common- you need 3 antigen negative cells that do not react (usually you have many more to rule out all of the other antibodies) and 3 antigen positive cells to rule in.  If you are calling multiple antibodies, you need 3 that are positive for EACH antigen but negative for all the others.  In your example, if you were to ID both anti-Fya and anti-C, you would need 

3 Fy(a-), C- that were negative

3 Fy(a+), C- that were positive

3 Fy(a-), C+ that were positive

 

Some labs only require 2 positive cells, but the 3 and 3 rule gives you 95% confidence in the ID.

 

I think ruling out on a single "homozygous" cell is OK, but I know there is a lot of debate around that.

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We use the 2 and 2 rule.

 

The way I understood the math is that 3 and 3 rule = 0.05 p value if you're running only 6 cells total. We use a three cell screen and an 11 cell first-line panel. You should be able to get an acceptable p value based on those numbers of cells tested. On that note, there's a lot of discussion about the relevance of p value in the first place.

 

Kanter, M. H., Poole, G. D. and Garratty, G. (1997), Misinterpretation and misapplication of p values in antibody identification: the lack of value of a p value. Transfusion, 37: 816–822.

 

We use one homozygous cell for rule outs, with some exceptions.

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I understand that 2 negs and 5 pos or vice versa gives the same statistical likelihood of the antibody specificity as 3 & 3 and is easier to get on some specificities, especially if there are multiple antibodies.  We rule out with a single double-dose cell except I like to see more than one to rule out Jka (could argue the same for Jkb, but anti-Jka is a much more common antibody).  We have some exceptions like ruling out C & E in the presence of anti-D is allowed with one single dose cell.

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We do the same thing as many others here with the 3x3 rule, with at least one homozygous cell for dosage-dependent. 

 

I appreciate the comment here about rule-outs with only one cell for all negative screen patients though!  Statistics aside, It seems like overkill to have to have 3 cells for r/o r/i s in many cases.

 

Scott

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We use 2 homozygous cells for rule outs. We make exceptions for C and E in the presence of anti-D and for K since finding those homozygous cells is not very feasible most of the time. The 2 cell rule out is what our reference lab uses. They ID with 2 antigen positive cells and 2 antigen negative cells. We still use 3 and 3.

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  • 2 weeks later...

From our antibody rule out policy:

If the pattern suggests:

1) a single antibody specificity, then rule out is based on a minimum of ONE negative homozygous cells;

2) multiple antibodies / autoantibodies, or there are ambiguous or unexplained reactions, then rule out is based on a minimum of TWO negative homozygous cells.

To be honest, I am not quite sure about the reason of TWO negative homozygous cells needed for point 2).

Not long ago, in order to meet that requirement, one of our technologists had to do selected cells from other two different panels to exclude two antibodies although the 1st panel already had one negative homozygous cells for each antibody. Does you lab have the same policy? Could you please explain the difference between 1) and 2)? Thank you.

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No, we do not follow such a policy (it may be unique to your facility!).

To address this issue operationally, I use the AntigenPlus program. http://www.antigenplus.com/

This program standardizes the rule-out process (excluding antigens to which the patient may have antibody). This is only the first of the many steps required for antibody identification.

Everyone in my staff of generalists is required to use the program.

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  • 6 months later...

This continues to be a stumbling block for techs. My predecessor allowed for rule-outs on heterozygous cells, which I do not like with the exception (big) K. I was taught that it is OK to rule out on a single homozygous expression when there is a negative reaction. 

I was also taught that to get p-values you had to have 1 positive and 7 negatives or 7 positives ad 1 negative OR 3 positives and 2 negatives or 2 positives and 3 negatives. I guess this is how we defined 'ruling in' although in my Med Tech program that verbiage was never used.

Is this defined as a recommendation from any authoritative/accreditation body or in the AABB Technical manual?

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AABB changed their algorhythm a few years ago to 2 pos and 2 neg cells to accommodate the IRLs since some abs/ags are so rare that it was impossible to find 3pos/neg cells (high/low incident ags).  The Technical Manual always had a p value chart for determining the p values of pos vs neg - it still exists on p400 of the latest version of the TM.   

 

Whether you r/o with homozygous, heterozygous depends on the rules your Medical Director wants you to use.  I learned you could r/o anything with one homozygous expression (we won't even think about the Duffy ags in black folks); heterozygous we could r/o if enzyme pretreated (obviously not those ags destroyed by the pretreatment) or run with PeG.  Sometimes you have to r/o with heterozygous cells - as cited above Big C/E with r'r or r"r.  One needs to remember that dosage does not mean heterozygous cells do not react, just that they react weaker than homozygous.

 

If you have techs making determinations based on their preferences rather than policy YOU have a problem! 

Edited by David Saikin
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  • 2 weeks later...

I see you are switching to the Echo.  I have noticed that dosage is the norm on this platform.  I have changed my rule out policy to be 2 homozygous cells to rule out C,c,E,e,Fya, Fyb, Jka, Jkb,M,N,S, and s.  We have almost missed a couple of kidds by only ruling out with 1 homozygous cell. 

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Without consulting my current antigrams, you are finding enough homozygous cells epfeiffer to do all these rule outs with select cells? How did you happen to pick up the Kidds- incompatible, Kidd pos unit crosmatches?

 

We did switch to the Echo back in September and things are OK with some significant exceptions that I continue to address, mostly non-specific allos (or are they false pos cells?) and equivocal results, most of which go with the non-specifid allos. . I am about to put up a new post on using pre-warmed plasma on the Echo for your reading pleasure everyone.

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