Validating saline replacement procedure, Uniform titer procedure

[Kathy]

My hospital currently does not have a SOP for the saline replacement procedure. How would I validate this? Also, we currently do anti-A, anti-B titers with room temperature incubation. How would we validate CAP's "Uniform Procedure", which includes an AHG phase?

[David Saikin]

For Saline replacement: what are you validating? You would need a specimen that displays rouleaux, one with an antibody and one with both (I guess).

I have reservations about CAPs uniform titer procedure (it is a 1:3 dilution in each tube - 1 volume of cells and 2 volumes of ab). IN Chemistry, that is a 1:3, I don't know why it is not in BB.

[Eagle Eye]

same here. I personally do not like this procedure. But I may have to implement it.

This is too cumbersom for component titer.

[nmartin]

I also have an issue with the CAP proficiency survey for titers. We report out both a RT and AHG result for Isohemagglutinin titers, CAP allows both RT and AHG results only if you use their uniform procedure. But since we use our own procedure, we can only report either RT or AHG.

I have written CAP but got no response from them.

Also their uniform procedure for Anti D doen't even match the method in the AABB technical manual which we have used for 30+ years. Where did they come up with their uniform procedures?

[Mabel Adams]

Not that I like the uniform titer procedure myself, but, David, isn't all blood bank testing a 1:3 dilution--2 drops plasma to one drop of cells? Both the tech manual and the uniform procedure just use measured volumes for more precision, I think.

My argument with it is that our lab does not have a defined w+ reaction and no such grade exists in our computer. I can't see the advantages compared to the work involved in getting everyone retrained.

[Eagle Eye]

Not that I like the uniform titer procedure myself, but, David, isn't all blood bank testing a 1:3 dilution--2 drops plasma to one drop of cells? Both the tech manual and the uniform procedure just use measured volumes for more precision, I think.

My argument with it is that our lab does not have a defined w+ reaction and no such grade exists in our computer. I can't see the advantages compared to the work involved in getting everyone retrained.

I am in same boat as you are....

[David Saikin]

Not that I like the uniform titer procedure myself, but, David, isn't all blood bank testing a 1:3 dilution--2 drops plasma to one drop of cells? Both the tech manual and the uniform procedure just use measured volumes for more precision, I think.

My argument with it is that our lab does not have a defined w+ reaction and no such grade exists in our computer. I can't see the advantages compared to the work involved in getting everyone retrained.

Mabel - not if you wash your cells to a dry button first - removes the saline/diluent from the equations. AND if they all are 1:3, then the titers shouldn't be reported in multiples of 2 (2,4,8,16 . . . ).

[Mabel Adams]

Do you do all tube testing on a dry button then, or only titers? I always assumed the dilutions 1:2 etc. were relative to neat. By your definition, those of us that don't use dry cell buttons are diluting the neat plasma. Also, I seem to remember that the net amount of antibody in the test is more important than whether it is diluted or not--at least up to a point. Back when, they used to try to come up with ways to concentrate antibody and then finally realized that they could just add 4 or 6 drops of sample instead. So, 4 drops of a 1:2 dilution of the sample puts the same amount of antibody into the tube as 2 drops of neat, right?

[David Saikin]

I only do titers in dry buttons . . . I will trust your math . . .

[Mabel Adams]

I am updating our titration procedure. As previously written it does not include making master dilutions but just makes serial dilutions of 100 microliters so that the entire dilution is used in the test. I was taught that smaller volumes of dilutions are less accurate so a master dilution is advised. Does anyone know how much difference this really makes? Does anyone else do it this way?

[Malcolm Needs ☆]

I am updating our titration procedure. As previously written it does not include making master dilutions but just makes serial dilutions of 100 microliters so that the entire dilution is used in the test. I was taught that smaller volumes of dilutions are less accurate so a master dilution is advised. Does anyone know how much difference this really makes? Does anyone else do it this way?

WE do - because it is more accurate.

[amym1586]

Would anyone be willing to share their saline replacement SOP? 

[goodchild]

Do you have a copy of the technical manual Amy? There's your best bet.

[AMcCord]

When I did titers, I did make a master dilution. I also think that it's more accurate/consistent.  Students could routinely replicate my results exactly from their master dilution. (Plus...if you have check cells that don't work , it's quicker to set the whole mess up again if you don't have to redo the dilutions.) I dumped titers quite a while back when the surveys got really pricey - I let the reference folks have all the fun. Always did hate that procedure for some reason.

[Dansket]

If the standard serial dilution process produced a 1+ or stronger result in the 1: 256 dilution tube, I would make a master dilution of 1:500 using a 50mL volumetric flask.  Subsequent dilution would be 1:1000, 1:2000, 1:4000, etc.

[Malcolm Needs ☆]

On 11/02/2016 at 1:36 PM, AMcCord said:

When I did titers, I did make a master dilution. I also think that it's more accurate/consistent.  Students could routinely replicate my results exactly from their master dilution. (Plus...if you have check cells that don't work , it's quicker to set the whole mess up again if you don't have to redo the dilutions.) I dumped titers quite a while back when the surveys got really pricey - I let the reference folks have all the fun. Always did hate that procedure for some reason.

I agree with ALL of what you say, including, as a Reference folk, the bit about hating titrations!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!